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Abstract:

A method of promoting differentiation of one or more human stem cells into
human coronary endothelial cells on at least one surface of a synthetic
tubular structure to be used to make a human hybrid carotid graft is
provided. The method includes arranging a plurality of human stem cells
on the synthetic tubular structure to yield a hybrid stem cell/synthetic
tubular structure and subjecting ex vivo, the hybrid stem cell/synthetic
tubular structure to three dimensional dynamic conditions effective to
promote differentiation of the one or more human stem cells into human
coronary endothelial cells on the at least one surface.

Claims:

1. A method of promoting differentiation of one or more human stem cells
into human coronary endothelial cells on at least one surface of a
synthetic tubular structure to be used to make a human hybrid carotid
graft, said method comprising:(a) arranging a plurality of human stem
cells on the synthetic tubular structure to yield a hybrid stem
cell/synthetic tubular structure; and(b) subjecting ex vivo, said hybrid
stem cell/synthetic tubular structure to three dimensional dynamic
conditions effective to promote differentiation of said one or more human
stem cells into human coronary endothelial cells on said at least one
surface.

2. The method of claim 1, wherein said at least one surface comprises an
inner surface of said synthetic tubular structure.

3. The method of claim 1, wherein said at least one surface comprises an
outer surface of said synthetic tubular structure.

4. The method of claim 1, wherein said human coronary endothelial cells
cover at least 50% of said at least one surface of said synthetic tubular
structure.

5. The method of claim 1, wherein said human coronary endothelial cells
cover at least 60% of said at least one surface of said synthetic tubular
structure.

6. The method of claim 5, wherein said at least one surface comprises an
inner surface of said synthetic tubular structure.

7. The method of claim 5, wherein said at least one surface comprises an
outer surface of said synthetic tubular structure.

8. The method of claim 1, wherein said human coronary endothelial cells
cover at least 70% of said at least one surface of said synthetic tubular
structure.

9. The method of claim 8, wherein said at least one surface comprises an
inner surface of said synthetic tubular structure.

10. The method of claim 8, wherein said at least one surface comprises an
outer surface of said synthetic tubular structure.

11. The method of claim 1, wherein said human coronary endothelial cells
cover at least 80% of said at least one surface of said synthetic tubular
structure.

12. The method of claim 11, wherein said at least one surface comprises an
inner surface of said synthetic tubular structure.

13. The method of claim 11, wherein said at least one surface comprises an
outer surface of said synthetic tubular structure.

14. The method of claim 1, wherein said human coronary endothelial cells
cover at least 90% of said at least one surface of said synthetic
tubular structure.

15. The method of claim 14, wherein said at least one surface comprises an
inner surface of said synthetic tubular structure.

16. The method of claim 14, wherein said at least one surface comprises an
outer surface of said synthetic tubular structure.

18. The method of claim 1, wherein said human coronary endothelial cells
cover 100% of said at least one surface of said synthetic tubular
structure.

19. The method of claim 18, wherein said at least one surface comprises an
inner surface of said synthetic tubular structure.

20. The method of claim 18, wherein said at least one surface comprises an
outer surface of said synthetic tubular structure.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a Continuation of PCT/US2006/045715, filed Nov.
30, 2006 and a Continuation-in-Part of U.S. application Ser. Nos.
11/440,152, filed May 25, 2006; 11/440,156, filed May 25, 2006, filed May
25, 2006; 11/440,155, filed May 25, 2006; 11/440,091, filed May 25, 2006;
11/440,158, filed May 25, 2006, which in turn are Continuations of U.S.
application Ser. No. 09/973,433, filed Oct. 8, 2001, now U.S. Pat. No.
7,063,942 and International Application No. PCT/US2001/042576, filed Oct.
9, 2001, now Publication No. WO 2002/032224 A1, which claim the benefit
of U.S. Provisional Application No. 60/239,015, filed Oct. 6, 2000. The
entire disclosure of the prior applications are considered as being part
of the disclosure of the accompanying application and are hereby
incorporated by reference therein.

BACKGROUND OF THE INVENTION

[0002]1. Field of the Invention

[0003]The invention relates to a method for making a human hybrid carotid
bypass graft and, more particularly, a method of promoting
differentiation of one or more human stem cells into human coronary
endothelial cells on at least one surface of a synthetic tubular
structure to be used to make a human hybrid carotid graft.

[0004]2. Background of the Related Art

[0005]Hemodynamics plays an obligate role on the function and phenotype of
vascular cells (i.e. endothelial cells, smooth muscle cells, fibroblasts,
etc.) and tissues in the cardiovascular system during disease and healthy
states. Cardiovascular disease is the leading cause of death in North
America, Europe and the developing world, with coronary heart disease and
atherosclerosis being amongst the most prominent cardiovascular diseases.
Atherosclerosis is a disorder in which the coronary arteries become
clogged by the build up of plaque along the interior walls of the
arteries, leading to decreased blood flow which can in turn cause
hypertension, ischemias, strokes and, potentially, death. Associated
systemic risk factors include hypertension, diabetes mellitus, and
hyperlipidemia, among other factors.

[0006]Atherosclerosis and other cardiovascular diseases, such as
peripheral arterial disease (PAD), occur regularly and predictably at
sites of complex hemodynamic behavior and, consequently, motivates
further investigation into the role of hemodynamics in cardiovascular
diseases. For example, atherosclerosis has been shown to occur in sites
of complex hemodynamic behavior. Surgical intervention is often employed
to treat it, and may include insertion of a balloon catherter to clean
out the plaquie, and insertion of a stent within the vessel to enable it
to remain open, or may include multiple bypasses of the clogged vessels.
Bypass surgery involves the removal of a section of vein from the
patient's lower leg, and its transplant into the appropriate cardiac
blood vessels so that blood flows through the transplanted vein and thus
bypasses the clogged vessels. A major problem associated with bypass
surgery is the patency of the vessels to be used in the bypass. The
bypass vessels are prone to failure, which may occur within a short
period of time after bypass surgery, or after a period of several years.
Hemodynamic forces have been implicated as a major factor contributing to
the failure of the bypass vessels.

[0007]Hemodynamic forces, which are forces generated by irregular flow,
and in particular, by the (sometimes irregular) flow of blood, are known
to have numerous influences on blood vessels, including, but not limited
to effects on blood vessel cell structure, pathology, function, and
development. In the specific example of blood vessel structure and
pathology, the vascular cells lining all blood vessels, endothelial cells
(ECs), are important sensors and transducers of two of the major
hemodynamic forces to which they are exposed. These forces include wall
shear stress ("WSS"), which is the fluid frictional force per unit of
surface area, and hoop stress, which is driven by the circumferential
strain ("CS") of pressure changes. Wall shear stress acts along the blood
vessel's longitudinal axis, while circumferential strain is associated
with the deformation of the elastic artery wall (i.e., changes in the
diameter of the vessel) in response to oscillation or variation in
vascular pressure. Wave reflections in the circulation and the inertial
effects of blood flow cause a phase difference, the stress phase angle
("SPA"), between CS and WSS. The SPA vanes significantly throughout the
circulation, and is most negative in disease prone locations, such as the
outer walls of a blood vessel bifurcation such as the carotid sinus and
the coronary arteries. Hemodynamic forces have been shown to dramatically
alter endothelial cell function and phenotype (i.e., higher shear stress
[low SPA] is associated with an atheroprotective gene expression profile,
and a low shear stress [large SPA] is associated with an atherogenic gene
expression profile).

[0008]ECs can influence vasoactivity and cause vessels to contract or
dilate depending on the blood flow (shear stress) and pressure (causing
stretch or CS), and thus are one component which is critical to blood
pressure regulation among the many important factors which influence
and/or are dependent on the hemodynamics. ECs are just one type of cell
which is directly influenced by hemodynamics. Numerous other cell types
may also directly or indirectly influenced by hemodynamics and mechanical
forces.

[0009]As discussed above, hemodynamic forces have been shown to
dramatically alter endothelial function and phenotype. For example, the
coronary arteries are the most disease prone arteries in the circulation
and have the most extreme SPA in the circulatory system, typically having
a large, negative value, yet do not have a particularly low shear stress
magnitude, thus suggesting that complex hemodynamic factors that include
the SPA are important in cardiovascular function and pathology.
Accordingly, there is a great need to study vascular biology in a
complete, integrated, and controlled hemodynamic environment, preferably
in 3-dimensions. However, to date, detailed knowledge of the
simultaneous, combined influence of the time varying patterns of WSS and
CS on EC biological response has not been technologically feasible.

[0010]More specifically, existing systems have focused on the individual
effects of either WSS or strain on ECs separately. The most common WSS
systems use a 2-dimensional stiff surface, such as, for example, a glass
slide, for the EC culture on the wall of a parallel plate flow chamber,
or a cone-and-plate type chamber, to simulate wall shear stress alone,
which is only one hemodynamic condition. In such a system, the WSS must
usually remain steady due to difficulties in simulating pulsatile flow,
and strain or stretch effects must be omitted. Further, cyclic straining
devices can only generate strain by stretching cells on a compliant
membrane, without flow, and typically only in 2-dimensions. Both types of
systems are obviously limited in the fidelity with which they can
simulate a true, complete hemodynamic environment.

[0011]To address the need for simultaneous pulsatile strain and shear
stress, a silicone tube coated with ECs was introduced. However,
simulators using these tubes could only achieve phase angles (SPA) of
about -90 degrees, if any, which is inadequate for simulating coronary
arteries (SPA>-180 or -250 degrees), the most disease prone vessels in
the circulation, or other regions of the circulation such as peripheral
circulation, carotid, renal, organ hemodynamics, or head and brain
hemodynamics, to name a few. A more complete physiologic environment
which provides time-varying uniform cyclic CS and pulsatile WSS in a
3-dimensional configuration over a complete range of SPA is sill needed.

[0012]Substantially all past research and development has focused only on
obvious, one-dimensional blood flow or shear stress hemodynamic force
characteristics, even though, based on physics, mathematics, and
experimentation, there are clearly a multitude of dimensions associated
with the with many simultaneous hemodynamic forces present in vivo, such
as pressure and strain. Physiologic environments are highly dynamic and
nonlinear, the cardiovascular system is certainly no exception. There is
a need to preserve 3-dimensional vascular geometry while simultaneously
and independently controlling hemodynamic forces such as, for example,
pressure, flow, and stretch, as well as many other parameters and forces)
in a cell and tissue culture environment in order to more fully and more
accurately recapitulate in vivo hemodynamic environments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]The invention will be described in detail with reference to the
following drawings in which like reference numerals refer to like
elements, wherein:

[0014]FIG. 1A is a schematic view of a system for recreating a hemodynamic
environment in accordance with an embodiment of the invention;

[0015]FIG. 1B is a schematic view of a reservoir for use with the system
shown in FIG. 1A;

[0016]FIGS. 2A-2E are schematic views of systems for recreating a
hemodynamic environment in accordance with embodiments of the invention;

[0017]FIGS. 3A-3D are schematic views of systems for recreating a
hemodynamic environment in accordance with embodiments of the invention;

[0018]FIGS. 4A-4D are schematic views of a chamber which may be applied
with any of the systems shown in FIGS. 2A-2E and 3A-3D;

[0019]FIGS. 5A-5C illustrate specimen shapes which may be applied with any
of the systems shown in FIGS. 2A-2E and 3A-3D;

[0020]FIGS. 6A-6E illustrate exemplary chamber(s)s with specimen(s)
mounted therein which may be applied with any of the systems shown in
FIGS. 2A-2E and 3A-3D;

[0021]FIGS. 7A-7D illustrate an exemplary mounting system which may be
applied with any of the systems shown in FIGS. 2A-2E and 3A-3D;

[0022]FIGS. 8A-8E illustrate a coupling system which may be applied with
any of the systems shown in FIGS. 2A-2E and 3A-3D;

[0023]FIGS. 9A-9B are flowcharts illustrating operation of the systems
shown in FIGS. 2A-2E and 3A-3D; and

[0024]FIGS. 10A-10H are graphs of pressure, diameter and flow rate
conditions generated by the systems shown in FIGS. 2A-2E and 3A-3D.

[0025]FIG. 11 shows a side view of a specimen in accordance with an
embodiment of the invention;

[0026]FIG. 12 shows examples of cross-sections of tubular structures
according to various embodiments of the invention;

[0027]FIG. 13A shows several examples for the measurement of the parameter
D(t);

[0028]FIG. 13B is a schematic cross sectional view of an example of a
multi-layer tubular structure;

[0029]FIG. 14 shows examples of tubular structures;

[0030]FIG. 15 shows multiple regions in an exemplary tubular structure
where dynamic conditions can be linked to global dynamic conditions
measured at the input and the output, respectively;

[0031]FIG. 16 shows an alternative block diagram of a system according to
another embodiment of the invention;

[0032]FIGS. 17A and 17B show examples of various forms or types of dynamic
conditions;

[0033]FIG. 18 shows examples of classes of dynamic conditions that can be
simulated according to various embodiments of the invention;

[0034]FIG. 19 shows a block diagram of a controller according to an
embodiment of the invention;

[0035]FIG. 20 shows a block diagram of a translator according to an
embodiment of the invention;

[0036]FIG. 21 shows an exemplary physiological coronary flow;

[0037]FIG. 22 shows an exemplary pressure/flow loop subsystem in
accordance with an embodiment of the invention;

[0038]FIGS. 23a-23d are diagrams that show various stages of a plurality
of pumps;

[0039]FIG. 24 shows a plurality of states during one cycle of operation;

[0057]FIGS. 44A-44B show exemplary embodiments of a prove sensor according
to an embodiment of the invention.

[0058]FIGS. 45A-45C and 46 show exemplary tubular structures in accordance
with embodiments of the invention;

[0059]FIGS. 47A-47G show exemplary tubular structures illustrating
exemplary second order dynamic conditions in accordance with embodiments
of the invention;

[0060]FIG. 48 show a flowchart of a process of matching second order
dynamic conditions to a selected target according to an embodiment of the
invention;

[0061]FIG. 49 shows a flowchart of a process for combining biologic and
non-biological materials according to an embodiment of the invention; and

[0062]FIG. 50 shows NO levels for cells experiencing different dynamic
conditions produced by an embodiment of a system according to the
invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0063]Any reference in this specification to "one embodiment," "an
embodiment," "example embodiment," "embodiments," etc., means that a
particular feature, structure, or characteristic described in connection
with the embodiment is included in at least one embodiment of the
invention. The appearances of such phrases in various places in the
specification are not necessarily all referring to the same embodiment.
Further, when a particular feature, structure, or characteristic is
described in connection with any embodiment, it is submitted that it is
within the purview of one skilled in the art to effect such feature,
structure, or characteristic in connection with other ones of the
embodiments.

[0064]A hemodynamic simulation system in accordance with embodiments of
the invention as embodied and broadly described herein overcomes current
technological limitations in biomedical research and, particularly, in
vascular research are overcome by physically reproducing both normal and
diseased physiologic states in a controlled environment. A precise and
complete physiologic environment is achieved via control of salient
dynamic conditions such as, for example, pressure, flow, and diameter,
that consequently control the predominant dynamic forces, WSS and CS.
This is achieved through independent control of these dynamic conditions,
thus allowing for independent control over a variety of dynamic
parameters and forces such as the magnitude and phase of the pulsatile
WSS and CS at a wide range of SPA. The system provides for the recreation
of real dynamic patterns, complex and simple, while also meeting the
stringent requirements for sterility and minimal media volume critical in
cell and tissue culture systems.

[0065]The system neatly integrates engineering and biological principles
by imposing a realistic, time varying mechanical environment on a test
specimen, such as, for example, living vascular cells, to provide a model
of normal and diseased cardiovascular function to help guide many areas
such as future therapeutic strategies, stem cell therapy, cell and tissue
regeneration or engineering, genetic or pharmacologic. The independent
control of pulsatile flow and pulsatile pressure to provide for
independent control over WSS, CS and pressure is a significant
breakthrough which, at first, seems paradoxical. That is, classically,
pressure and flow are coupled. However, in a dynamic oscillatory or
sinusoidal environment such as is present in this system, flow and
pressure can be independently controlled in a variety of ways to achieve
the desired result. [DRM--Where do we describe the possible fluid
component compositions??]

[0066]FIG. 1A is a schematic view of a system for reproducing a
hemodynamic environment and, more particularly, a schematic view of a
flow loop of such a system, in accordance with one embodiment of the
invention as broadly described herein. In this system 1, flow of fluid
and/or media is initiated by a steady flow system 30 and introduced into
a flow loop, where it passes into a specimen unit 10. An individual or
multiple specimen 12 may be positioned in the specimen unit 10 by a
mounting system 80. The single/multiple specimen 12 are exposed to fluid
and/or media carried by the fluid, as well as to the dynamic environment
produced by the system 1. The specimen unit 10 may be coupled, and
preferably detachably coupled, to the flow loop by a coupling system 300.

[0067]Dynamic pressure and flow conditions within the specimen unit 10 may
be generated and maintained by a pressure/flow control system 200, which
acts on the fluid traversing through the flow. Fluid may be substantially
continuously recirculated through the flow loop for a required amount of
time/cycles, or based on another such controlling parameter which would
govern the flow through the flow loop. In other embodiments, a
predetermined amount of fluid/media may be introduced into the flow loop
and held in the specimen unit 10 for a predetermined amount of
time/cycles, or other such controlling parameter, as the pressure/flow
control system 200 generates the required conditions in the specimen unit
10.

[0068]The action of the steady flow system 30 and the pressure/flow
control system 200 may be controlled by a control system 70. The control
system 70 may also receive data related to various parameters from
various sensors positioned throughout various portions of the system 100,
such as, for example, the specimen unit 10, the steady flow system 30,
the pressure/flow control system 200, and other locations along the flow
loop. In certain embodiments, the control system 70 provides for dynamic
control of the system 1 based on feedback provided by a variety of
sensing/detection systems (not shown in detail in FIG. 1A). In
alternative embodiments, the control system 70 may simply operate the
system 100 in accordance with a previously stored algorithm based on
conditions desired in the specimen unit 10 and/or throughout the flow
loop, without feedback.

[0069]FIG. 1B is a schematic view of a reservoir 20 that may be optionally
used in the steady flow system 30 shown in FIG. 1A. The reservoir 20 may
hold fluid for initial and re-circulation, and may allow media to be
introduced into or siphoned from the flow loop. That is, as fluid/media
is returned to the reservoir 20, a portion, or all of the fluid/media may
be redirected, or siphoned off and not recirculated. For this purpose,
the reservoir 20 may be partitioned into inflow 20a and outflow 20b
portions, or the siphoned fluid may be diverted to a holding tank or
other such vessel or flow system (not shown).

[0070]The reservoir 20 may further include a sampling port 21 which
samples incoming fluid before recirculation and/or diversion to the
outflow portion 20b or a holding tank. The sampling port 21 may be
adapted to divert incoming fluid based on, for example, its measurement
of parameters such as, for example, concentration of media components,
contamination levels, circulation time/cycles and the like. Likewise, the
incoming portion 20a of the reservoir 20 may include an inflow port 22 to
allow for the introduction of additional fluid and/or media as required,
and may include sensors 23 linked to the control system 70 which
continuously monitor levels/quantity of such fluid/media as it is
introduced into the flow loop.

[0071]The reservoir 20 may also include a port to atmosphere (not shown),
preferably with a sterile filter to preclude contamination from the
atmosphere. Additionally other cell or tissue types may be positioned
throughout the system, such as, for example, near the reservoir 20 or a
port thereof. For example, a chamber (not shown) containing hepatocytes
may be positioned in the flow loop so as to be exposed to the fluid in
the flow loop, as well as to at least some of the dynamic conditions in
the flow loop, if desired. This type of exemplary setup can be used to
provide other useful data such as, for example, drug metabolism data.

[0072]The positioning and interconnection of the components of the system
1 shown in FIG. 1A is merely exemplary in nature, and intended simply to
illustrate the presence of these components and their respective
functions within the system 1. Thus, for example, although the steady
flow system 30 shown in FIG. 1 is positioned adjacent the reservoir 20
and the pressure/flow control system 200, followed in the flow loop by a
portion of the coupling system 300, it is well understood that the steady
flow system 30 may include various components positioned throughout the
system 1 to provide the capabilities required of the steady flow system
30. Likewise, although the pressure/flow control system 200 is shown
simply on an ingress side of the specimen unit 10, it is well understood
that the pressure/flow control system 200 may include various components
positioned throughout the system 1 to fulfill the requirements of the
pressure/flow control system 200. Such reasoning applies to the remaining
components of the system 1, including the coupling system 300, mounting
system 80, control system 70, and specimen unit 10, as will be better
understood from the following discussion.

[0073]FIG. 2A is a schematic view of an exemplary system 1000 for
reproducing a hemodynamic environment, in accordance with one embodiment
of the invention as broadly described herein. Although the specimen unit
10 shown in FIG. 2A includes a chamber 11, which forms an enclosure for a
single specimen 12, the system 1000 may also include a single chamber 11
housing a plurality of specimens 12, a plurality of chambers 11 each
housing a single specimen 12, a plurality of chambers 11 each housing a
plurality of specimens 12, and a plurality of chambers 11, some housing a
single specimen 12, and some housing a plurality of specimens 12, as will
be further described below in connection with FIGS. 6A-6C. Further, as
shown in FIGS. 6D-6E, the system 1000 may also include an
individual/multiple specimen 12 not surrounded by any type of enclosure
or chamber 11. Instead, an individual/multiple specimen 12 may be aligned
directly with the flow loop.

[0074]In alternative embodiments, the chamber 11 may be jacketed 15, as
shown, for example, in FIG. 4B, thereby enabling circulation of a cooled
or heated fluid through the chamber 11 and specimen 12, in order to
maintain the temperature required by the specimen 12 and an associated
trial. Alternatively, the chamber 11 may be immersed in a water bath 16
at an appropriate temperature, as shown, for example, in FIG. 4C, or may
include a conditioned circulation path 17, as shown, for example, in FIG.
4D to achieve the desired temperature control effects.

[0075]The system 1000 may generally be run at a temperature of
approximately 37 degrees Centigrade, but can be operated at temperatures
ranging from approximately 20 degrees Centrigrade to approximately 50
degrees Centigrade, or whatever temperature may be required for a
particular trial.

[0076]**The specimen 12 may take many forms. In certain embodiments, the
specimen 12 may be a substantially tubular type, compliant structure made
of materials such as, for example, silicone, collagen, PTFE, fibrin, and
other such appropriate materials, which is lined with a variety of
cellular compounds and/or cells, such as, for example, endothelial cells
or stem cells on a fibronectin matrix used to simulate a vessel wall a
non-rigid tube that contains mammalian cells, a blood vessel excised from
a mammal, or other biocompatible substrate containing cells or onto which
cells can be grown or attached thereto. In other embodiments, the
specimen 12 may be a portion of an actual vessel (ex vivo), such as, for
example artery or vein, which is to be subjected to the hemodynamic
environment produced by the system 1000. Likewise, while the specimen 12
discussed herein are, simply for ease of discussion, substantially
tubular, the specimen 12 may also have an irregular form to more
accurately represent an actual physiological condition or environment,
such as, for example, a bifurcation a curve, physiologic vascular
segment, or changes in cross section to reproduce a constriction present
in an actual vessel. Samples of some specimen 12 which have such
irregular forms are shown in FIGS. 5A-5C.

[0077]The specimen 12 may include various entities, such as, for example,
different cell types. These may be cells other than vascular cells which
may be attached or integrated in the specimen 12, or which may be
non-attached and circulating, such as, for example, immune cells, such
as, for example, leukocytes, monocytes, and the like, stem cells, such
as, for example, adult, embryonic, progenitor, and the like, cancer
cells, red blood cells, platelets, and other such cell types. Other organ
cells such as, for example, hepatocytes for liver toxicity assessment or
adsorption distribution metabolism excretion (ADME) examination, may also
be incorporated into the system for activities such as, for example,
testing and screening purposes. Similarly, numerous different components
may be added to the media to simulate different conditions, including,
but not limited to, cholesterol for hyperchloesterolimia, growth factors
for growth and development, calcium for vulnerable plaque and lesion
formation, and other such components.

[0078]In the system 1000 shown in FIG. 2A, the steady flow system 30
includes a reservoir 20 and a steady flow pump 30a. Fluid which is to be
introduced into the specimen unit 10 may be drawn out of the reservoir 20
by the steady flow pump 30a which initiates and maintains a substantially
constant, substantially uniform flow of fluid from the reservoir 20 into
the flow loop. Other types of pumps or components which may be used to
initiate and maintain such a steady flow may also be appropriate. The
steady flow pump 30a shown in FIG. 2A is disposed between the reservoir
20 and the specimen unit 10, at a position upstream from an ingress 10a
into the specimen unit 10. However, the steady flow pump 30a may also be
disposed at other positions within the system 1000, based on the type of
component(s) used to generate the steady flow from the reservoir 20, as
well as the placement of other components of the system 1000.

[0079]The desired test environment may be developed and maintained within
the specimen unit 10 by the pressure/flow control system 200. In the
system 1000 shown in FIG. 2A, the pressure/flow control system 200 may
include a first pressure/flow control 40 positioned upstream of the
ingress 10a into the specimen unit 10 and a second pressure/flow control
50 positioned downstream of an egress 10b from the specimen unit 10. In
alternative embodiments, the system 1000 may also include a third
pressure/flow control 60 which further controls an internal pressure
and/or flow within the specimen unit 10, and/or an external pressure. The
first, second and third pressure/flow controls 40, 50 and 60 may be
combined as necessary during operation of the system 1000, depending on
which conditions are to be reproduced in the specimen unit 10 and which
properties are to be monitored/studied during a particular trial. For
example, the third pressure/flow control 60 may not be required in some
situations, such as, for example, when a specimen 12 is aligned directly
with the flow loop, without a chamber 11 surrounding the specimen, as
shown in FIGS. 6D-6E, or when there is a chamber 11 in use but all the
required trial conditions can be reproduced with, for example, just the
first and second pressure/flow controls 40, 50, as shown in the
embodiment of the system 2000 shown in FIG. 2B. Preferably, the
pressure/flow control system 200 includes at least a first pressure/flow
control 40 and a second pressure/flow control 50.

[0080]Conditions throughout the flow loop, including within the specimen
unit 10 and/or those experienced by the specimen 12 itself, may be
controlled and monitored by a control system 70. The control system 70
may include a processor (not shown in detail) which substantially
continuously transmits parameters to be monitored and data to be gathered
from at least one, and preferably a plurality of sensors provided at
various positions within the flow loop. FIG. 2A shows an exemplary
placement of sensors, in which a sensor 72 is provided proximate, and
preferably within, the specimen unit 10, a sensor 74 is provided upstream
of the specimen unit 10, between the ingress 10a to the specimen unit 10
and the first pressure/flow control 40, and a sensor 76 is provided
downstream of the specimen unit 10, between the egress 10b of the
specimen unit 10 and the second pressure/flow control 50.

[0081]The plurality of sensors may serve a variety of functions. For
example, the sensor 72 may be a sensor which monitors a condition of the
specimen 12, such as, for example, a size/diameter, growth rate or wall
thickness of the specimen 12, or a condition of the fluid/media
surrounding the specimen 12, such as, for example, concentration of
components of fluid/media, or a diffusion of fluid/media (water flux) or
of solutes (i.e. fluorescent labeled LDL or dextran, components of the
fluid/media) through an outer wall of the specimen 12. Other functions
for a so positioned sensor may also be appropriate. Likewise, the sensors
74, 76 may be, for example, sensors which measure a pressure and/or flow
rate at a corresponding position in the flow loop. Other types of sensors
and/or sensor placement may also be appropriate. Data collected by the
plurality of sensors may be used by the control system 70 to adjust
operation/control parameters for the steady flow system 30, the
flow/pressure control system 200, and the like. Arrangement of any number
and type of sensors may be varied as appropriate based on the control
requirements and data gathering needs dictated by a particular trial.

[0082]Numerous types of sensors and actuators may be used to gather the
data required by the control unit 70. For example, wireless
nanotechnology, microelectromechanical systems (MEMS), or electrochemical
based systems may be integrated at various points within the system 1000
to detect and transmit data such as, for example, real time metabolite
and proteins present, % absorption and absorption rates, pressure, flow,
and other such parameters. This type of technology may also be used as a
vehicle to deliver a fluid, cells, or chemicals, such as a drug, to a
specifically targeted area of the specimen 12, to transmit images from a
specific area, or to take other types of readings from a specific area of
the specimen 12 as required. Ultrasound technology may be used to monitor
flow rates, dissipation/diffusion rates, growth rates, and the like.
Strain gauges may be used to monitor pressure/pressure fluctuations
throughout the flow loop. The numerous sensors and actuators can be
placed in numerous locations throughout the system 1000, including both
the overall system flow loop and the external flow loop (including the
chamber 11).

[0083]Other appropriate sensing systems may include, but are not limited
to, laser detection systems, and optical detection systems such as, for
example, fluorometers, luminometers, or microscopes. These systems could
also include probes to measure cell and/or layer integrity on the
specimen 12, and/or to apply electrical stimuli to the specimen 12. For
example, electrical stimuli may be applied directly to the specimen 12 at
various locations such as, for example, at a mounting point to measure
cell layer integrity or enhance growth, function or the like. Various
other numbers, types and relative positioning of sensors may also be
appropriate, depending on the particular conditions to be reproduced, and
the amount and type of parameters to be monitored and the data to be
gathered.

[0084]The first, second and third pressure/flow controls 40, 50, 60 may
take many forms. For example, as shown in FIG. 2A, the first and second
pressure/flow controls 40, 50 may be, for example, pumps connected to the
flow loop upstream and downstream of the specimen unit 10, and the third
pressure/flow control 60 may be an external pressure/flow control system
connected to the specimen unit 10 to exert an external pressure on the
specimen 12, or to control a flow of fluid in the chamber 11, such as,
for example, the radial flow of fluid through the outer circumferential
walls (transmural flow and transport) of the specimen 12. In this
example, respective drive units (not shown) of the first, second and
thirds pressure/flow controls 40, 50, 60 are preferably independently
controlled. In certain embodiments, the pumps 40, 50 may be piston-type
pumps, such as, for example, bellows pumps, which can be independently
varied in oscillatory motion with typical waveform parameters such as
magnitude and phase to produce a desired overall effect in the specimen
unit 10.

[0085]Preferably, any oscillatory waveforms or signals can be programmed
into such pumps which may be used in the first and second pressure/flow
controls 40, 50. These oscillatory waveforms or signals may include, but
are not limited to, for example, a blood pressure waveform, a blood flow
waveform, a diameter waveform, a sinusoidal waveform, a saw-tooth
waveform, a square waveform, a frequency control, a slew rate, a duty
cycle, a period, a percent systolic or diastolic, harmonic frequencies,
magnitude, phase, and the like. Other parameters may also be appropriate
for programming into these exemplary pumps which may be used in the first
and second pressure/flow controls 40, 50 depending on the effect desired
in the specimen unit 10.

[0086]This control of magnitude and phase, amongst other features
mentioned above, in the pertinent parameters provides simulation of a
wide range of precise and controlled hemodynamic parameters such as WSS,
CS, pressure, and the SPA, including in the range in which the most
diseased prone coronary arteries fall (SPA>-250 deg). In other
embodiments, the first and second pressure controls 40, 50 may include
valves, and preferably occluder valves, which are controlled by the
control unit 70 to control the flow there through in order to produce
similar effects. Since the flow which runs through the flow loop, and,
consequently, through the specimen 12, is related to wall shear stress
(WSS), and the pressure exerted on the specimen 12 is related to the
circumferential strain (CS), the pulsatile WSS and the pulsatile CS may
be independently controlled and thus may be uncoupled within a certain
range.

[0087]In alternative embodiments in which the pressure/flow control system
200 includes a third pressure/flow control 60, the third pressure/flow
control 60 may provide for numerous different, additional conditions to
be reproduced in the specimen unit 10, and thus may take numerous
different forms. For example, in certain embodiments, the third
pressure/flow control 60 may be an external pressure/flow control used in
combination with a chamber 11 surrounding one or more specimen 12. This
may include an external flow loop 59 which runs partially through the
chamber 11, as shown in the embodiment of the system 3000 shown in FIG.
2C, or may include a pump 65, such as the piston or bellows type pumps
discussed above with respect to the first and second pressure controls
40, 50, used to apply an external pressure to the specimen 12 within the
chamber 11, as shown in the embodiment of the system 4000 shown in FIG.
2D. Alternatively, the third pressure/flow control 60 may be a
combination of an external pump 65 and an external flow loop 59, as shown
in the embodiment of the system 1000 shown in FIG. 2A.

[0088]This external flow loop 59 may facilitate the introduction and/or
extraction of media from the chamber 11, or may be used to induce flow
and/or circulation in a particular direction within the chamber 11, such
as, for example, radially, such that the specimen 12 experiences
conditions such as, for example, expansion in a radial direction, bending
or other longitudinal deformation, or accelerated or decelerated
diffusion of media through the specimen 12 wall, or facilitate the
generation of other conditions within the chamber 11 as appropriate. As
shown in FIG. 2A and in more detail in FIG. 4A, the external flow loop 59
and external pump 65 may be combined to form the third pressure/flow
control 60.

[0089]An exemplary external pressure/flow control is shown in more detail
in FIG. 4A. In this embodiment, the third, or external pressure/flow
control 60 includes an external flow loop 59 coupled to the chamber 11
to, for example, induce a circulatory, oscillatory, or pulsatile flow or
pressure in the chamber 11, and/or to introduce additional media into or
extract media from the chamber 11. Tills exemplary third, external
pressure/flow control 60 may include an external steady flow unit 62 to
initiate and maintain flow through the external flow loop 59. The flow
through the external flow loop 59 may be a simple recirculation of fluid
in the chamber 11. Alternatively, the external flow loop 59 may include
its own reservoir 64 to hold, for example, media to be introduced into
the chamber 11. The external flow loop 59 may also include a varying flow
unit 66 to generate variations in the flow introduced into the chamber
11, such as, for example, a concentrated flow in a particular portion of
the chamber 11, or a pulsatile flow to further simulate actual dynamic
conditions. The varying flow unit may be a single piston or bellows type
pump, or may be pairs of pumps which operate similar to the first and
second pressure/flow controls 50, 60 described above. This type of
external flow loop 59 may be combined with a separate external piston or
bellows type pump 65 which may be separately coupled to the chamber 11,
or, alternatively, which may be incorporated into the external flow loop
59, to introduce additional forces as discussed above with respect to the
first and second pressure/flow controls 40, 50.

[0090]Numerous different algorithms and methodologies may be applied in
controlling the first, second and/or third pressure/flow controls 40, 50,
60 to produce a desired condition in the specimen unit 10 and/or
throughout the flow loop. For example, assuming, simply for purposes of
discussion that the steady flow system 30 is a steady flow pump 30a, and
the first and second pressure controls 40, 50 each include piston/bellows
type pumps, as in the systems shown in FIGS. 2A-2E, and the third
pressure/flow control 60 includes an external pump 65, control of the
various pumps may be coordinated to produce a desired condition. If the
pumps maintain a mechanical connection through, for example, an
adjustable cam that was able to control the timing or phase between the
external pump and the downstream pump, the external pump would operate at
a certain magnitude, as would the downstream pump, but they may peak at
different times. Likewise, the pumps may be coordinated
electromechanically to control there respective timing and phase or
synchrony. Thus, pressure/flow, whether it be upstream, downstream, or
external, may be controlled by the coordinated action of the pumps at
their respective location(s).

[0091]The specimen 12 is preferably positioned within the specimen unit 10
using a mounting system 80. The mounting system 80 may be used to
appropriately position the specimen 12, whether a chamber 11 is used or
not. Any number/type of mounting systems may be appropriate, depending on
the parameters and characteristics to be reproduced in the specimen unit
10, the properties of the specimen 12 to be studied during operation of
the flow loop, and the number of specimen 12 to be positioned in the
specimen unit 10. It is also useful and important to be able to reproduce
physical forces) such as, for example, axial strain, torsion and bending
forces, which may be present in an actual physiological environment on
the specimen 12 in the specimen unit 10, whether the specimen 12 is
contained in a chamber 11 of the specimen unit 10, or is simply coupled
to the flow loop through the mounting system 80.

[0092]As shown in FIGS. 7A-7D, in certain embodiments, fixed ends of the
specimen 12, which may be, for example, a silicone tube, an expanded PTFE
(ePFTE) tube, artery, vein, tissue engineered artery, and the like, may
be attached to a rigid tube 14 that can rotate about its longitudinal
axis. The tube 14 and/or specimen 12 are preferably sized so as to
correspond to the actual vessel which it is intended to simulate. For
example, in certain embodiments, the tube 14 and/or specimen 12 is
preferably between approximately 0.5 mm and 30 mm in diameter and various
lengths ranging from 1 cm and 80 cm (typically used in cardiovascular
surgery). In other embodiments, the specimen 12 may be a 2D substrate
such as a glass slide or other 2D silicone membrane structured
appropriate to that which it is to simulate. Again, the chamber 11 may or
not be present.

[0093]As shown, for example, in FIG. 7A, in certain embodiments the tube
14 may be attached to a mount 16 that is coupled to a carriage 18,
allowing the mount 16 to translate in the longitudinal direction. The
embodiment shown in FIG. 7A includes a carriage 18 at each end of the
specimen unit 10, and either one or both carriages 18 may move at a
particular time. However, only one carriage 18 may be necessary,
depending on conditions required in the specimen unit 10.

[0094]A coupler 15 may be attached to both the tube 14 and the mount 16 to
provide for independent movement within a predetermined range of motion.
The coupler 15 may then be attached to a drive system 17 such as, for
example, a linear actuator that imposes oscillatory or sinusoidal motion,
a stepper motor, an electrodynamic transducer, and the like, to provide
for motion in accordance with the prescribed conditions to be reproduced
in/by the specimen unit 10. More particularly, as shown, for example, in
FIG. 7B, gears 11 and racks 13 may be used to provide linear or torsional
motion of the tube 14 and/or specimen 12, with the racks 13 directing
motion to a corresponding gear 11 to turn the mount 16, to which the tube
14 and/or specimen 12 is attached, about its longitudinal axis. A gear 11
may also be used to move a rack 13 to translate the mount 16 in an axial
direction.

[0095]Preferably, the tube 14 has two ends, an upstream and a downstream
end, and either or both ends may experience controlled axial strain
and/or torsion. Alternatively, one end may remain fixed and experience no
motion, while the other end experiences some prescribed motion. More
specifically, the carriage(s) 18 may translate so as to draw the two
opposite ends of the tube 14 and/or specimen 12 apart to induce an axial
force, such as, for example, a component of axial stretching or strain.
The carriage(s) 18 may also translate so as to draw the two opposite ends
of the tube 14 and/or specimen 12 towards each other so as to induce a
force such as, for example, compressor or bending in the tube 14 and/or
specimen 12. A mounting of the tube 14 and/or specimen 12 in the specimen
unit 10 using the mounting system 80 is shown in FIG. 7C.

[0096]The oscillatory axial strain can be reproduced either with both
fixed ends of the tube 14 and/or specimen 12 oscillating, or with one
fixed end constant and the other end oscillating. The mean axial strain
or fixed end(s) of the rube 14 and/or specimen 12 may also be adjusted.
That is, variation in axial strain can remain constant, while the mean
axial strain or fixed end position is slowly increased. Torsion may be
achieved with both fixed ends of the tube 14 and/or specimen 12 rotating,
or with one of the two fined ends held constant. In one embodiment, the
tube 14 to which specimen 12 may be attached may be connected to a gear
13, that provides torsion driven by a rack gear 11. Although the rotation
angle can proceed to 360 degrees, the rotation angle is preferably
limited to avoid buckling. Preferably, the rotation angle is limited to 0
degrees±45 degrees with both fixed ends rotating in opposition, or to
0 degrees±90 degrees with one fixed end held in a constant position. A
relationship between axial strain and torsion can be simulated and varied
independent of each other.

[0097]Although the exemplary mounting system 80 shown in FIGS. 7A-7C
relies on the drive system 17 described above to provide the movement
necessary to generate axial strain, bending, and/or torsion, other
mechanisms which allow for control of the time varying position of the
ends of the tube 14 and/or specimen 12 may also be appropriate. Likewise,
although only one tube 14 and/or specimen 12 is shown mounted using the
mounting system 80 shown in FIGS. 7A-7C, it would be well understood that
the mounting system 80 could be readily adapted to receive multiple tubes
14 and/or specimen 12, as shown in FIG. 7D. In addition to the
longitudinal strain and torsion in or about the X axis, as described
above, in certain embodiments the mount positions may move in 3
dimensions, Y and Z) so as to rotate about the respective axes, Y and Z.

[0098]This mounting system 80, which in some embodiments may be considered
a hemodynamic axial strain and torsion simulator, may be incorporated
into a flow loop as described herein to reproduce additional hemodynamic
forces not reproduced by the various pumps and pressure/flow controls of
the system so as to provide a more complete physiological hemodynamic
environment. An embodiment of the system 5000 incorporating this
hemodynamic axial strain and torsion simulator shown in FIG. 2E.

[0099]In certain embodiments, the mounting system 80 can include
additional components such as additional drive systems 17 coupled to
either or both ends of tube 14 to provide longitudinal strain (e.g.,
stretch) and torsion (e.g., twist) along the Y axis. Alternatively, such
components may be directly or indirectly coupled to the specimen 12 or
tubular structure 1112 to controllably provide Y axis longitudinal
stretch and/or twist.

[0100]In additional embodiments, the mounting system 80 can include
additional components such as additional drive systems 17 coupled to
either or both ends of tube 14 to provide longitudinal strain and/or
torsion along the Z axis. Alternatively, such components may be directly
or indirectly coupled to the specimen 12 or tubular structure 1112 to
controllably provide Z axis longitudinal stretch and/or twist.

[0101]Accordingly, embodiments of the specimen holder 10 or pressure flow
loop subsystem 1105, for example using the mounting system 80 or
components directly or indirectly coupled to specimen 12, can provide
stretch and twist in single or opposite directions along individual or
combinations of the X, Y and Z axis of specimen 12 or tubular structure
1112. Embodiments according to the invention can locate such strain and
twist along the X, Y and Z axis at positions intermediate to ends of
tubular structure 1112 (e.g., region A), at branches of specimen 12
(e.g., FIGS. 5C, 5D and XI) or for multiple specimens 12 coupled in
series or parallel in specimen holder 10.

[0102]In certain embodiments, the specimen unit 10 may further include
additional components to further modify the flow therein when the
above-described components cannot achieve the desired result on their
own. Such additional components may include, for example, jets or
internal fins which could effect helical or secondary flow within the
chamber 11 as necessary, or be positioned in the flow loop such that as
the fluid enters the chamber 11, the fluid flow is substantially helical.

[0103]Alternative methods or components can be used to generate
substantially helical flow, circular flow or wave reflections in specimen
12 or tubular structure 1112. In certain embodiments, systems 1, 5000,
1101 can be mounted on mechanical systems that rotate (e.g.,
horizontally) at a fixed distance around a center point combined with
vertical movement relative to the center point. Such controlled circular
and vertical motion (e.g., merry-go-round) of the systems 1, 5000, 1101
can controllably generate a helical flow of fluid in conduit 3701,
specimen 12 or tubular structure 1112. Further, in certain embodiments,
the rotation around and vertical movement relative to the center point
can be at a steady or time varying speed (e.g., constant speed,
increasing speed, pulsed speed, sinusoidal speed or the like). Additional
movement of the systems 1, 5000, 1101 can be provided by varying the
distance of the systems from the center point in a controlled fashion.
Thus, additional embodiments can selectively provide one or more of these
individual or reciprocal movements (e.g., tangential, vertical, radial or
in combinations thereof) of the system 1, 5000, 1101 around a center
point to generate controlled fluid dynamics (e.g., dynamic conditions)
according to the viscosities of the fluids and tubular structures
therein.

[0104]In certain embodiments, a coupling system 300 may be used to couple
the specimen unit 10 to the flow loop. Although the coupling system 300
is not required in order for the system 1 to operate as described herein,
the coupling system 300 may, for example, allow for quick disconnect of
the specimen unit 10, and may be adapted to accommodate a specimen unit
10 which includes a chamber 11 with either single or multiple specimen
12, or may accommodate a specimen unit 10 including a single or multiple
specimen 12 without a chamber 11. The coupling system 300 may also
facilitate the removal and replacement of specimen unit(s) 10 while
maintaining necessary sterility of the remainder of the flow loop. The
coupling system 300 may also allow for quick removal for post-processing
of the specimen(s) 12 for further analysis and the like.

[0105]An exemplary coupling system 300 is shown in FIGS. 8A-8E. The
coupling system 300 includes a first coupler 310 which may be separably
coupled to a second coupler 320 to form a coupling unit 330. Preferably,
the coupling system 300 includes a coupling unit 330 (i.e., set of first
and second couplers, 310, 320) positioned on opposite ends of the
specimen unit 10 such that the specimen unit 10 may be removed from the
flow loop by separating each second couplet 320 from its corresponding
first coupler 310. In such an embodiment, the first coupler 310 remains
connected to a portion of the flow loop, while the second coupler 320
remains coupled to a portion of the specimen unit 10.

[0106]In certain embodiments, the first and second couplers 310, 320 may
include corresponding inter-engaging protrusions (male) and recesses
(female) which couple the first and second couplers 310, 320 by, for
example, snap fit, or other such means which would facilitate easy
engagement and disengagement while maintaining seal and sterility
integrity. When the corresponding inter-engaging protrusions and recesses
are engaged, their respective through holes are aligned so as to allow
fluid to pass therethrough. Upon disengagement of the first and second
couplers 310, 320, flow inhibitors, such as, for example, simple disc
valves (now shown) inhibit the flow of fluid therethrough, thereby
maintaining seal and sterility integrity when separated as well.

[0107]It is well understood that any such position and number of
corresponding protrusions and recesses would be appropriate, depending on
a number of specimen 12 to be sampled and other such considerations.
Likewise, although the exemplary first and second couplers shown in FIGS.
8A-8E are rectangular in shape, it is well understood that a shape of the
first and second couplers 310 and 320 and the positioning and number of
the associated protrusions and recesses may be adapted to suit the needs
of a particular application. Thus, in this exemplary coupling system 300,
the fluid/media in the flow loop may be supplied to the coupling system
300, and particularly, to the coupling unit 330 positioned upstream of
the specimen unit 10, and split so as to supply fluid/media to twelve
specimen 12. Likewise, if multiple specimen units 10 each with its own
coupling unit 330 at its ingress 10a and egress 10b are aligned with the
flow loop, one and/or all of the specimen unit(s) 10 may be removed and
replaced without compromising critical features such as, for example,
system integrity or sterility.

[0108]As discussed above, it is preferable that a coupling unit 330 be
positioned on each end of the specimen unit 10. The first coupler 310
includes a number of protrusions 312 extending from a first side 311
towards its respective end of the flow loop. In the coupling unit 330
positioned upstream of the specimen unit 10, these protrusions 312 are
coupled to flow loop supply lines which receive fluid/media from the
reservoir 20. In the coupling unit 330 positioned downstream of the
specimen unit 10, these protrusions 312 are coupled to drain lines
entering from the downstream side of the specimen unit 10. The second
side 313 of the first coupler 310 includes a corresponding number of
recesses 314 which engage corresponding protrusions 322 formed on a first
side 321 of the second coupler 320. The protrusions 322 are fit into the
recesses 314, and an o-ring 319 may be used to improve a scaling
characteristic therebetween. The second side 323 of the second coupler
320, which preferably faces the specimen unit 10, includes a number of
corresponding protrusions 324 which extend toward the specimen unit 10
and specimen(s) 12 positioned therein so as to supply fluid/media thereto
or drain fluid/media therefrom.

[0109]Thus, fluid/media from the flow loop passes through the first and
then the second coupler 310, 320 of the upstream coupling unit 330, and
then passes through the specimen unit 10, where the specimen(s) 12 are
exposed to the fluid/media. The fluid/media is drained out of the
specimen unit 10 and passes into the second coupler 320 and then first
coupler 310 of the coupling unit 330 positioned on the downstream end of
the specimen unit 10, where it is introduced back into the flow loop. In
alternative embodiments, the second side 323 of the second coupler 320
may be used when individual chambers 11 and/or specimen(s) 12 are to be
disengaged from the flow loop while others are to remain connected, such
as, for example, during time series analysis, where different chamber(s)
11 and/or specimen(s) 12 must be disengaged at different points in time
during a trial to provide sample data for progression type analysis.

[0110]Although the coupling units 330 are shown at upstream and downstream
ends of the specimen unit 10, the quick disconnect/reconnect qualities
and commensurate preservation of sterility afforded by these types of
coupling units 330 may also be useful at numerous other locations
throughout the flow loop. For example, a set of coupling units 330 may be
positioned on opposite ends of the first pressure/flow control 40 or the
second pressure/flow control 50 so as to make these systems modular and
easily removable/replaceable as well.

[0111]The various components of the systems 1, 1101 described above may be
joined to form the flow loop using, for example, tubing. This tubing
generally comprises any suitable type of laboratory tubing which is
capable of being sterilized, including silicone tubing, or other
comparable laboratory or medical-surgical tubing. The distances between
the various components and the corresponding length of the tubing may be
chosen so as to minimize the total volume of fluid used. Preferably,
these lengths are calculated to provide a maximum flow rate, and to avoid
turbulence in the system, based upon boundary layer theory, as known to
those skilled in the art. Generally, it is preferable to minimize the
amount of fluid used in order to reduce the costs of media utilization,
drug treatment, and cell by-product (such as, but not limited to,
proteins, metabolites and like) detection and the like.

[0112]Systems for reproducing a hemodynamic environment in accordance with
other embodiments of the invention as broadly described herein will now
be discussed with respect to FIGS. 3A-3D. The systems and combinations of
components discussed above with respect to the embodiments of the system
shown in FIGS. 2A-2E are readily adapted to the embodiments shown in
FIGS. 3A-3D. Thus, for example, the coupling system 300, mounting system
80, and control system 70 as described above may each be applied to the
systems as shown in FIGS. 3A-3D. Thus, there are any number of possible
combinations of these components, as well as their placement within
embodiments of the system, and, simply for ease of discussion, any
duplicative description is omitted.

[0113]The system 6000 shown in FIG. 3A includes a specimen unit 10 with a
specimen 12 mounted therein by a mounting system 80 and coupled to a flow
loop by coupling units 330. A reservoir 20, first pressure/flow control
40 and second pressure/flow control 50 cause fluid/media to flow from the
reservoir 20 through the flow loop as described above. However, steady
flow from the reservoir 20 through the first and second pressure/flow
controls 40, 50 is now provided by a steady flow system 30 comprising a
pair of upstream and downstream pressure/flow control occluders 35-38
provided upstream and downstream of the specimen unit 10 which provide
for steady flow of fluid/media into the flow loop and appropriate flow
into and out of the first and second pressure/flow controls 40, 50

[0114]These pressure/flow control occluders 35-38, which may be, for
example, pinch valves, or flow occluders and the like, positioned
upstream and downstream of the specimen unit 10 occlude flow and pressure
in a controlled oscillatory manner, thus allowing for steady or mean flow
without a steady flow pump.

[0115]In operation, when one occluder per pressure/flow control 40 or 50
is open, the other is preferably closed. Thus, for example, when the
first upstream occluder 35 is open, the second upstream occluder 37 is
closed and pump 40 can eject or push fluid toward the open occluder 35
which is connected to the specimen 12 at an appropriate pulsatile or
other such rate as dictated by a required condition. Likewise, to fill or
supply the pump 40 occluder 37 is open while occluder 35 is closed,
allowing pump 40 to draw fluid from the reservoir 20 through the open
occluder 37, where it may be held by the pump 40 and closed occluder 37
until, for example, sufficient fluid has been collected therein to
operate the pump 40 to create the particular flow dictated by the desired
condition. The downstream occluders 36, 38 operate in a similar manner.
This allows for control of various hemodynamic parameters such as flow,
pressure, and diameter and consequent hemodynamic forces in the specimen
unit 10.

[0116]Alternatively, a variety of conditions may be achieved while
maintaining a mean pressure by controlling the first pressure/flow
control 40 and upstream occluders 35, 37 along with the second
pressure/flow control 50 and downstream occluders 36, 38 to essentially
maintain a mean pressure while still permitting control of flow and
pressure. One exemplary manner in which this may be achieved is by
closing upstream occluder 37 and opening upstream occluder 35. This will
allow fluid to move toward the specimen unit 10 and pressure and flow
will continue to increase in the specimen unit 10 until downstream
occluder 36 is opened and 38 is closed (or open), thus allowing fluid to
exit the specimen unit 10 and reducing pressure accordingly. As pressure
and flow reach the desired value, upstream occluder 35 may be closed, and
upstream occluder 37 may be opened. This allows a mean pressure and flow
to be maintained in the specimen unit 10 through appropriate, coordinated
timing of the opening and closing of the occluders 35-38.

[0117]A system for reproducing a hemodynamic environment in accordance
with another embodiment of the invention as broadly described herein is
shown in FIG. 3B. The system 7000 shown in FIG. 3B is similar to the
system 6000 shown in FIG. 3A. However, the system 7000 includes a third
pressure/flow control 60 which includes an external flow loop 59
separately coupled to the specimen unit 10 as described above. However,
the external flow loop 59 shown in FIG. 3B obtains steady flow in the
external flow loop 59 from a pair of external pressure/flow control
occluders 68, 69 (rather than an external steady flow pump). Likewise,
the systems 8000 and 9000 shown in FIGS. 3C and 3D are similar to the
system 7000 shown in FIG. 3B. However, the system 8000 includes an
external pressure control 65 as discussed above, in combination with an
external flow loop 59 which now includes another pair of external
pressure/flow control occluders 61 and 63. This additional pair of
external pressure/flow control occluders 61, 63 may be employed to
further maintain constant pressure or flow in the chamber 11 if so
desired. In the system 9000 shown in FIG. 3D, the third pressure/flow
control 60 is simply an external pump 65 externally coupled to the
chamber 11.

[0118]As can be well understood, the various means set forth herein may be
combined as necessary and expedient to achieve a desired result. Thus,
for example, steady flow may be provided both in the flow loop and in the
external flow loop by a number of different component(s) and/or
combination(s) of components, such as, for example, a steady flow pump,
or a pairing of pressure/flow occluders and their operation with a
corresponding pressure/flow control or pump. Likewise, a third, external
pressure control may or may not be included in the pressure/flow control
system, and may include, for example, simply an externally applied
pressure/flow control in the form or a pump, or a partial or full
external flow loop, or a combination thereof. The coupling system and
mounting system discussed above may be applied to any of the combinations
of components as appropriate/required to provide enhanced utility and/or
ease of use.

[0119]Likewise, any of these systems may include a variety of other
components not shown in detail in these particular figures, such as, for
example, a flow damper, or noise filter, that reduces vibrations or noise
in the fluid flow. Resistors, such as flow restrictors or clamps that
restrict or reduce flow, may be used to increase pressure in the specimen
unit 10 or other location within the flow loop if the resistor is
appropriately positioned, such as downstream of the downstream pump if
this condition is desired in the specimen unit 10. Capacitors, such a
chamber that has air and fluid in it and acts as a compliance chamber,
can be placed upstream or downstream of the specimen unit 10, preferably
downstream, to help adjust various hemodynamic parameters such as the
impedance between flow and pressure.

[0120]The various system components, such as, for example, tubing,
reservoir(s), and pumps, may be made of a variety of materials. In
certain embodiments, these components may be made from disposable
materials such as, for example, plastic, polypropylene, PETG, and the
like to facilitate providing and maintaining a sterile environment, as
well as ease of set up and change out of test trials. In other
embodiments, these components may be made of non-disposable materials,
such as, for example, metals, to provided enhanced durability, structural
integrity, and the like. In still other embodiments, these components may
be made of a combination of disposable and non-disposable materials, that
can be sterilized by, for example, ETO, autoclave, gamma irradiation, and
the like, such materials preferably being non-toxic materials.

[0121]An exemplary operation of the systems shown in FIGS. 2A-2E and 3A-3D
will now be discussed with reference to FIGS. 9A-9C. As shown in FIG. 9A,
first, the specimen unit 10 is coupled to the flow loop (S100),
preferably using the coupling units 330 as described above. The steady
flow system 30 is activated to draw fluid/media from the reservoir 20,
which is holding fluid and/or media therein, into the flow loop (S200),
and the pressure/flow control system 200 is also activated (S300) so that
as the fluid/media is drawn through the upstream coupling unit 330 and
into the specimen unit 10, the appropriate dynamic conditions are present
in the specimen unit 10. Alternatively, the pressure/flow control system
200 may be activated first, followed by the steady flow system 30, or the
two systems may be activated simultaneously, depending on the
requirements of a particular trial. The introduction of the fluid/media
into the specimen unit 10, and particularly the characteristics of the
fluid/media associated with pressure and/or flow, as well as the
conditions within the specimen unit 10, and particularly those associated
with pressure and/or flow of the fluid/media in the specimen unit 10, are
established by the pressure/flow control system 200 based on parameters
preset in the control unit 70. As the specimen 12 experiences the dynamic
conditions reproduced in the specimen unit 10, the sensors collect data
and transmit the data to the control system 70 for monitoring and
analysis (S400). The control unit 70 may dynamically monitor, control,
and adjust the operation of the steady flow system 30 and the
pressure/flow control system 200 as necessary based on its substantially
continuous analysis of the data collected.

[0122]The fluid/media then passes out of the specimen unit 10, again, at a
pressure and/or flow condition established by the pressure/flow control
system 200 based on control parameters in the control system 70. The
outgoing fluid/media passes through the downstream coupling unit 330 and
back towards the sampling port 21 of the reservoir 20. At the sampling
port 21, the fluid/media is directed to either the reservoir 20, an
outflow portion 20b of the reservoir 20, or a holding tank outside the
flow loop, again based on preset parameters stored in the control system
70 and characteristics measured by the sensor 23.

[0123]The system 1000 continues to operate in accordance with the control
parameters set by/in the control system 70 until a preset condition or
parameter is reached (S500). The governing parameter or condition, which
may be preset in the control system 70, may be, for example, time/elapsed
time, cycles, a remaining level of fluid and/or media in the reservoir
20, a concentration or other characteristic of the fluid/media as it is
returned to the sampling port 21 of the reservoir 20, and other such
parameters and/or conditions. When the preset condition has been
satisfied, the steady flow system 30 and the pressure/flow control system
200 are deactivated (S600, S700), the control system 70 collects and
analyzes any remaining data as required (S800), the specimen unit 10 is
decoupled from the flow loop (S900) and post-processing analysis is
performed. When other conditions are included, such as, for example,
axial stretch and/or torsion components provided by the mounting system
80, these auxiliary systems may be activated as necessary after the flow
conditions are set. The sensors can initiate sensing as required to
either provide feedback or no feedback to the control system 70
throughout operation of embodiments of the system as required.

[0124]As discussed above, the processor 70 may be used to control the
various components of embodiments of the system to produce a desired
condition or set of conditions in the specimen unit 10 and/or at various
locations throughout the flow loop. The control system 70 may control
embodiments of the system to operate in numerous modes, including, for
example, a first mode in which the control system 70 controls embodiments
of the system based on manually entered or preset parameters/algorithms,
with little to no feedback from various sensors which may be positioned
throughout the flow loop, and no commensurate dynamic adjustment (an open
loop control mode). The control system 70 may also control embodiments of
the system in a second mode in which the manually entered or preset
parameters/algorithms may be dynamically adjusted based on feedback
received from the numerous sensors positioned throughout the flow loop (a
closed loop control mode). Feedback may include, for example, pressure,
flow, diameter, strain, metabolite production, and other such
measurements related to a particular condition/set of conditions.
Numerous other parameters may also be monitored and fed back to the
control system 70 to provide for the dynamic adjustment of the control
parameters and algorithms applied by the controller based on the
parameters dictated by a particular condition/set of conditions. Other
control modes, including a combination of the open and closed loop
control modes, may also be appropriate. These control modes are discussed
in more detail below.

[0125]FIG. 9B is a flow chart of the operation of the controller
throughout the process shown in FIG. 9A, in accordance with an embodiment
of the invention. It is assumed that at least one, and preferably a
plurality of dynamic conditions and associated control
parameters/algorithms producing the consequent hemodynamic forces are
previously stored in a memory portion (not shown) of the control system
70 for selection by an operator at the initiation of a particular trial.
In alternative embodiments, conditions and/or control parameters may be
selected or entered manually. Such manually entered conditions/parameters
may include, for example, flow magnitude, pressure, magnitude, phase
relation) and other such parameters which may produce a desired
hemodynamic condition.

[0126]As shown in FIG. 9B, first a hemodynamic condition/set of conditions
is selected (S10). The control parameters/algorithms associated with a
selected condition/set of conditions may be retrieved from a previously
stored set of control parameters/algorithms (S30), or may be manually
entered (S25), based on requirements dictated by a particular trial and
other such considerations (S20). For example, a specific hemodynamic
region in which certain flow and pressure conditions will have certain
associated wall shear stresses and circumferential strain levels may be
chosen to produce a patient specific condition, such as, for example, a
distressed coronary artery with a typical large phase difference between
pressure and flow, or a healthy condition in which a phase difference
between flow and pressure is relatively small. As discussed above, these
conditions and associated control parameters may be previously stored in
the control system 70. Likewise, parameters such as flow magnitude,
pressure magnitude, phase relation, and the like may be manually entered,
and then resulting conditions calculated by the control system 70, if
desired.

[0127]Once the control parameters/algorithms have either been retrieved
from memory (S30) or manually entered (S25), the control system 70 sends
the corresponding control parameters/algorithms to the various affected
components (S40) such as, for example, the steady flow system 30, the
first second and third pressure/flow controls 40, 50, 60 and their
corresponding components which are included in the pressure/flow control
system 200, the mounting system 80 to provide for appropriate axial
strain and/or torsion, and any other components linked to the flow loop
which should be controlled in a given manner to produce the selected
hemodynamic condition/set of conditions. These control parameters may
include, for example, output voltages or currents with appropriate
oscillatory patterns (such as, for example, sinusoids or blood pressure
waveforms) to produce the desired conditions.

[0128]As the control system 70 operates embodiments of the system, it
determines whether or not feedback has been received (S50). If no
feedback has been received from the sensors, the control system 70 checks
to see if any new/additional control parameters have been manually
entered (S55). If new control parameters have been entered (S25), the new
control parameters are received by the control system 70 and transmitted
to the components (S40). If new control parameters have not been entered,
the control system 70 can determine if the trial is complete (S70), and,
if not, continues to transmit the valid control parameters to system
components (S40). This process continues until the control system 70
determines that the trial is complete (S70).

[0129]If feedback is received from the sensors (S50), the control system
70 determines if adjustment to the control parameters is required based
on the feedback (S60). To accomplish this, the control system 70 may, for
example, conduct a comparison of the control parameters as originally
established to a set of measured parameters. Alternatively, the control
system 70 may receive the various feedback parameters, and perform a
calculation to determine actual dynamic conditions at a particular
location compared to conditions which were initially established for that
location. If, based on these comparisons/calculations, the control system
70 determines that no adjustment is required, the control system 70 then
determines whether the trial is complete (S70), and, if not, continues to
transmit the valid control parameters to the system components (S40). If
feedback is received from the sensors (S50) and adjustment of the control
parameters is required based on the comparisons/calculations, then the
control parameters are adjusted (S65) and the adjusted control parameters
are transmitted to the system components (S40). This process continues
until the control system 70 determines that the trial is complete (S70).

[0130]As set forth above, the various embodiments of the system described
herein may be adapted to receive numerous different types of specimen and
be operated and configured in a variety of different manners based on the
requirements dictated by a particular trial. For illustrative purposes,
operation of the system 2000 shown in FIG. 2B, in which a compliant
specimen including, for example, a compliant silicone tube lined with
endothelial cells so as to be representative of an actual vessel,
in-vivo, with similar mechanical properties such as, for example, modulus
of elasticity, compliance, and the like, has been mounted in the specimen
unit 10 for drug screening and testing will now be discussed in more
detail. It is well understood that this is just one example of the many
applications of each of the various systems set forth herein, and is not
meant to in any way be construed as so limiting the application or
operation of embodiments of the system as embodied and broadly described
herein.

[0131]If, for example, the silicone tube lined with endothelial cells
discussed above is to be subjected to a particular hemodynamic condition
for testing, appropriate parameters are set to produce such a condition.
In this example, a healthy hemodynamic condition may be represented by a
WSS of 10±10 dynes/cm2 at a pressure of 70±20 mmHg and a
circumferential strain represented by a change in diameter of ±4%,
yielding an SPA of 0 degrees at a frequency of 1 Hz. As discussed above,
these control parameters may be manually entered, or they may be stored
in a memory portion of the control system 70 in association with a given
hemodynamic condition, and accessed as necessary prior to the initiation
of a trial.

[0132]Once the appropriate hemodynamic condition is selected and the
corresponding control parameters are made available, the control system
70 controls to the steady flow pump 30a to operate to initiate a
circulation of fluid through the flow loop. The first and second
pressure/flow controls 40, 50, which, in this example, are likely to be
bellows pumps, oscillate to produce oscillatory waveforms corresponding
to the required dynamic conditions. This may be accomplished by) for
example, the first pressure/flow control 40, considered in this example
to be the upstream pump, creating an increase in flow and pressure
directed toward the specimen unit 10, while the second pressure/flow
control 50, considered in this example to be the downstream pump,
simultaneously creating an increase in flow and pressure directed toward
the specimen unit 10. The coordinated action of the upstream and
downstream pumps and the resultant pressure and flow conditions produced
in the specimen unit 10 result in an oscillatory component at or above
the 0 degree SPA associated with a healthy hemodynamic condition for the
such a specimen.

[0133]The oscillatory waveforms generated by the coordinated action of the
upstream and downstream pumps in this example may be varied by varying
the action of the upstream and downstream pumps accordingly. Thus, for
example, rather than directing an increase in pressure and/or flow toward
the specimen unit 10, one of both of the upstream and downstream pumps
may instead operate to draw fluid collected in the specimen unit 10 out
of/away from the specimen unit, thereby producing a differentiated effect
on the specimen mounted therein. In this particular example, if bellows
pumps are employed at the upstream and downstream positions, this may be
accomplished by allowing the bellows portion of the pumps to fill with
fluid from the flow loop through the action of the steady flow pump 30a
which maintains a mean flow through the flow loop concurrent with the
action of the upstream and downstream pumps, and then controlling a
release of fluid from the bellows toward the specimen unit as required to
produce the desired effect. Or, alternatively, the bellows may be filled
from the specimen unit 10 side of the respective pump and the release of
the collected fluid into the flow loop controlled to produce an
alternately directed effect.

[0134]As described above, in this particular example, the steady flow pump
30a maintains a mean flow throughout the flow loop, concurrent with the
action of the upstream and downstream pumps. Thus, as the upstream and
downstream pumps collect and discharge fluid toward/away from the
specimen unit 10, at least some, if not all of the fluid running through
the pumps as they operate is replenished with circulating fluid. As fluid
leaves the downstream pump, it travels toward the reservoir 20, where, in
this particular example, a portion thereof is periodically siphoned off
at the sampling port 21 for sampling. The remainder of the fluid is then
returned to the reservoir 20 for recirculation in this particular
example, although, as discussed above, in other applications, this return
fluid may be fully or partially diverted to an outflow portion 20b or
holding tank rather than recirculated. This recirculation of fluid and
operation of the various pumps as described above is continued in
accordance with the established algorithms until a preset stop condition
is achieved. In an example such as this, in which a specimen is
undergoing drug testing, this stop parameter is often time based, i.e.,
exposure of the specimen 12 to a particular set of conditions for a given
amount of time, based on actual interaction of such drugs in-vivo.
However, as discussed above, this stop condition may vary based on
requirements dictated by a particular trial.

[0135]This is just one example of how one of the embodiments of the
invention may be employed for a drug screening and testing trial on a
compliant silicone tube lined with endothelial cells. It is well
understood that the various other components described herein may also be
applied to embodiments of the system to augment the capability of that
system and provide further variation in the dynamic conditions to which a
specimen may be exposed. For example, addition of a third pressure/flow
control 60, which may include a pressure/flow control pump, a full
external flow loop, or a combination thereof, may provide for further
variation of the flow environment created within the specimen unit 10 and
commensurate additional combinations of hemodynamic force. The addition
of torsion and/or axial strain through implementation of the capabilities
of the mounting system 80 may further expand the sets of condition(s)
which may be created in the specimen unit 10 and experienced by a
particular specimen. Numerous different environments and parameters may
be monitored and/or control algorithms adjusted based on a number, type
and placement of a variety of sensors throughout the selected system and
the capabilities of the control system 70.

[0136]FIGS. 10A-10H provide graphical representations of the various
stress (WSS) and strain (CS) conditions which may be achieved by the
various systems FIGS. 2A-2E and 3A-3C, graphically depicted in terms of
pressure (P), diameter (D) and flow rate (Q). More specifically, FIGS.
10A-10H demonstrate control of magnitude, phase, and frequency of flow,
pressure, and diameter waveforms in a specimen 12 such as, for example an
artificial or silicone artery, and the unique conditions that may be
achieved by the system 100 in the chamber 11. The various conditions and
combinations of conditions graphically depicted in FIGS. 10A-10H are
tabulated in Table 1 below.

[0137]In Table 1, an oscillator condition for one of the parameters Q, P
or D is shown as True or "T" state, while a constant or non-time varying
condition is shown as a False or "F" state. For example, a condition in
which there is oscillatory flow Q (True state) with no change in pressure
P or diameter D (False state) as shown in line C of Table 1 and
graphically depicted in corresponding FIG. 10C may now be achieved due to
the capabilities provided by the combination of components provided in
the systems shown in FIGS. 2A-2E and 3A-3C. Further, a condition in which
there is oscillatory flow Q (True state), oscillatory diameter D (True
state), and no change in pressure P (False state) as shown in line D of
Table 1 and graphically depicted in corresponding FIG. 10D may now be
achieved due to the capabilities provided by the combination of
components provided by the systems shown in FIGS. 2A-2E and 3A-3C.

[0138]FIG. 11 shows a side view of a specimen, shown as a tubular
structure 1112, in a specimen unit (not shown) in accordance with an
embodiment of the invention. Specimen 1112 is represented as a tubular
structure having a length L'. Specimen 12, described above, includes, but
is not limited to, a tubular structure 1112. As used herein, tubular
structure 1112 includes any three dimensional structure capable of
passing fluid from one location to another. This includes shapes of any
section found in the cardiovascular system in humans or animals or any
shapes of sections, including but not limited to C, I, T, Y of FIGS.
5A-5D and 11. Tubular structures 1112 further include any shapes of
sections found in humans or animals that serve to transfer or pass fluid
from one location to another. For example, tubular structures can
include, but are not limited to, aortas, arteries, arterioles,
capillaries, venules, veins, vena cavas, pulmonary arteries and pulmonary
veins. Tubular structures can further be synthetic, partially porous,
permeable, grooved, microgrooved, hybrid biological/synthetic and/or
electrospun.

[0139]Region A as shown in FIG. 11 represents a portion or subsection of
specimen or tubular structure 1112. Specimen 1112 has a diameter of
approximately D(t) over a length L which is ≦L'. In accordance
with one embodiment of the invention, a sample has pressure P and flow Q,
if the measured pressure P and flow Q are substantially within ΔP
and ΔQ of the values of P and Q over the Region A. Hence, region A
represents a portion of tubular structure 1112 in which pressure is
substantially between P±ΔP/2, flow is Q±ΔQ/2, and
diameter is D±ΔD/2, and a specimen is said to have dynamic
conditions P, Q and D, if the measured values of P, Q and D over a region
A are substantially within the ratios ΔP/PRange,
ΔQ/QRange and ΔD/DRange, respectively, where
PRange, QRange and DRange can be, for example, mean values
of the potential ranges of pressure, flow and diameter for specimen 1112.
In preferred embodiments, ΔP/PRange≦0.35, and
preferably ΔP/PRange≦0.25, and more preferably
ΔP/PRange≦0.15 and even more preferably
ΔP/PRange≦0.05, similarly
ΔQ/QRange≦0.35, and preferably
ΔQ/QRange≦0.25, and more preferably
ΔQ/QRange≦0.15 and even more preferably
ΔQ/QRange≦0.25, and similarly
ΔD/DRange≦0.35, and preferably
ΔD/DRange≦0.25, and more preferably
ΔD/DRange≦0.15 and even more preferably
ΔD/DRange≦0.05.

[0140]FIG. 12 shows examples of cross-sections of tubular structures 1112
according to various embodiments of the invention. The cross-sections of
tubular structures can be circular, ovular or elliptical, even lobe
shaped (such as a figure eight). Other embodiments of the invention may
include, tubular structures having a nearly two dimensional flattened
ribbon shape with an ovular and/or rippled shaped cross-section as shown
in FIG. 12.

[0141]In a preferred embodiment of the invention, specimen 1112 is not
completely rigid in that the shape of its cross-section may vary in
response to sufficiently large variations in dynamic conditions such as
pressure P(t), flow Q(t) will structures WSS, circumferential strain
(CS), stretch or Length (L), twist/torque (T) and so forth. Hence,
tubular structures preferably have at least some flexibility in the sense
that the diameter D(t) (as generally defined herein) can vary in response
to sufficiently large variations in pressure P(t), flow Q(t), stretch or
Length (L), and/or twist/torque (T) along a selected direction of
measurement.

[0142]FIGS. 12 and 13A demonstrate how diameter D(t) as used herein can be
a parameter generally indicative of the shape of a cross-section of
tubular structures. The shape of the tubular cross-section may be
non-circular, such as elliptical or ovular, in which case the diameter
D(t) represents a parameter indicative of variations in that
cross-sectional shape. For example, parameter D(t) can represent the
inner diameter, the outer diameter, the tubular structure's wall
thickness along one or more directions of the cross-sectional area along
a selected direction as shown in FIG. 12. The selected direction of
measurement can be in any direction with respect to the cross-sectional
area.

[0143]FIG. 13A shows several examples of how a direction of measurement
can be selected for the measurement of the parameter D(t) as well as how
the measurement of the inner and/or outer diameter of a cross-section of
tubular structures can be accomplished according to alternative
embodiments of the invention. For example, FIG. 13A shows parameters
D1 and D'1 representing the inner and outer diameter,
respectively, of a cross-sectional area of a tubular structure as
measured along the direction 1. Similarly, parameters D2 and
D'2 represent the inner and outer diameter, respectively, of the
tubular structure as measured along the direction 2. The parameter D(t)
can be combinations of D1, D'1, D2, and/or D'2. For
example, parameter D(t) might be the thickness of the walls of the
tubular structure along direction 1, namely, D1(t)-D'1(t).
Also, diameters can be measured along additional directions and those
values combined by controller 70 and/or independently monitored by
controller 70 as independent feedback signals.

[0144]The tubular structure may also be a multi-layer structure, in which
case parameter Dxy can represent the inner diameter of layer x along
direction y and D'xy can represent the outer diameter of layer x
along direction y.

[0145]FIG. 13B is a schematic cross sectional view of a human blood vessel
(e.g., artery or vein), which is an example of a multi-layer tubular
structure as broadly defined herein, which shows how the parameter D(t)
inner and/or outer diameters of a cross-section of multi-layer tubular
structures can be accomplished according to alternative embodiments of
the invention. Arteries and veins follow substantially the same
histological makeup. The inner most layer is an inner lining called the
endothelium, followed by a second layer of subendothelial connective
tissue. This is followed by a third layer of vascular smooth muscle,
which is highly developed in arteries. Finally, there is a fourth layer
of connective tissue called the adventitia, which contains nerves that
supply the muscular layer, as well as nutrient capillaries in the larger
blood vessels.

[0146]Parameters D11 and D'11 represent the inner and outer
diameters, respectively, of a cross-sectional area of a first layer of a
multi-layer tubular structure as measured along the direction 1.
Similarly, parameters D21 and D'21 represent the inner and
outer diameters, respectively, of a second layer of the multi-layer
tubular structure as measured along the direction 1.

[0147]Parameters D12 and D'12 represent the inner and outer
diameters, respectively, of a cross-sectional area of the first layer of
the multi-layer tubular structure as measured along the direction 2.
Similarly, parameters D22 and D'22 represent the inner and
outer diameters, respectively, of the second layer of the multi-layer
tubular structure as measured along the direction 2.

[0148]Tubular structures are further categorized into those which respond
to dynamic conditions in a substantially consistent manner and those that
do not.

[0149]Referring to FIG. 14, dynamic conditions gi(t) can be measured
at various locations on systems, in accordance with embodiments of the
invention. Feedback FBi(t) can include signals indicative of dynamic
conditions at locations other than regions A of specimen 1112. Such
dynamic conditions gi(t) might, from time to time, be referred to as
system or global dynamic conditions.

[0150]As used herein, stable dynamically responsive tubular structures are
tubular structures whose dynamic conditions at region A are substantially
repeatable for a given set of global dynamic conditions and/or local
dynamic conditions for a system 1101 according to embodiments of the
invention.

[0151]A system can be trained using a first tubular structure with a
stable and dynamic responsivity. If the relationship between the physical
structure of the first and any subsequent tubular structures is known and
these subsequent tubular structures have a stable dynamic responsivity,
then global dynamic conditions can be translated by controller 1103 to
yield dynamic conditions at the subsequent tubular structures a priori.
For example, if a system 1101 is trained using a first tubular structure,
and global dynamic conditions (e.g., FBi(t)) and input information
has been linked (in accordance with, for example, FIG. 29), then a second
stable tubular structure with an outer wall thickness twice that of the
first tubular structure, but the same inner diameter of the first tubular
structure could be inserted in specimen unit 10 of pressure/flow loop
subsystem 1105. Controller 1170 can then perform the appropriate
translations to yield local dynamic conditions at a corresponding region
A of the second tubular structure, provided the responsivity of a second
tubular structure with twice the wall thickness is known a priori.
Translation of properties between stable tubular structures can be
ascertained as known to those skilled in the art, for example, fluid
dynamics and fluid mechanics.

[0152]As discussed above, sensors, detectors, transmitters, receivers
and/or transceivers (referred to herein from time to time as "sensors")
can be arranged within, on and/or around pressure/flow loop subsystem
1105 to sense, detect and/or measure various dynamic conditions at
various locations in pressure/flow loop subsystem 1105 and/or to transmit
information. The locations of such sensors will yield feedback signals
FBi(t) corresponding to types of dynamic conditions (examples of
which are shown in FIGS. 17A and 17B) that could be considered global
dynamic conditions of system 1101. As with a single region A, known
variations of stable tubular structures can be linked to global dynamic
conditions by in controller 1103 in accordance with embodiments of the
invention.

[0153]If a given controller 1103 is trained using a stable tubular
structure having an architecture, for example, C, I, T, Y or some other
architecture, then controller 1103 may include processing which maps
those global dynamic conditions to multiple or alternative regions A for
a given tubular structure in accordance with embodiments of the
invention. For example, using a stable tubular structure, multiple
regions A1-A5 can be selected can be selected during a training process
and dynamic conditions at their respective locations can be linked to
global dynamic conditions measured at the input ( Gin) and the
output ( Gout) located at the input and output of specimen holder 10,
respectively, as shown in FIG. 15.

[0154]These system sensors include sensors, transmitters, receivers,
detectors, transceivers, etc., and can sense, detect, measure, transmit
and/or receive information which can be directly or indirectly associated
with dynamic conditions at any location. System sensors can be as small
or smaller than nanosensors or be large or more sophisticated systems,
such as an MRI, PET or other systems, as will be discussed herein.

[0155]FIG. 16 shows an alternative block diagram of a system 1101 with a
specimen or tubular structure 12, such as system 1 of FIG. 1A according
to another embodiment of the invention. System 1101 includes a controller
1103 and a pressure/flow loop subsystem 1105. Controller 1103 receives
input data or information corresponding to desired dynamic conditions and
translates that information to a set of N control signals fj(t),
j=1, 2, 3, . . . , N. The set of N control signals fj(t) can be a
single control signal or multiple control signals. Pressure/flow loop
subsystem 1105 includes pressure/flow loop components such as the various
elements, devices or subsystems in the embodiments discussed herein.
Pressure/flow loop components can include, for example, pressure/flow
control system 200 (FIG. 1A) and any elements, devices or subsystems
contained therein as well as other elements, devices or subsystems
contained in the embodiments of the systems discussed herein, such as
steady flow systems, specimen units, sensors, reservoirs, pumps, upstream
or downstream pumps, steady flow pumps, occluders, external pressure
controllers, axial strain system, slider carriage, torsion systems and
any other elements in the pressure/flow control systems. Control signals
fj(t) in turn are input to pressure/flow loop subsystem 1105 where
each control signal fj(t) controls and/or adjusts one or more of the
pressure/flow loop components.

[0156]As discussed above, the set of control signals fi(t) can be a
single control signal or multiple control signals for driving the various
components of the pressure/flow loop subsystem 1105. The various
pressure/flow loop components of the pressure/flow loop subsystem 1105
can be controlled with respective control signals fj(t). In one
embodiment, controller 1103 outputs a separate control signal fj(t)
for each component to be controlled in the pressure/flow loop subsystem
1105.

[0157]Alternatively, some or all of the components that make up the
pressure/flow loop subsystem 1105 could be mechanically coupled, such
that an adjustment to one component using a control signal fj(t)
will cause a predetermined adjustment in another component via such a
mechanical coupling. This allows for the adjustment of multiple
components using fewer control signals fj(t) than the number of
components in the pressure/flow loop subsystem 1105. Such mechanical
couplers are described in related U.S. Pat. No. 7,063,942 filed on Oct.
9, 2001, and incorporated by reference in its entirety.

[0158]In addition, the individual components in the pressure/flow loop
subsystem 1105 can also be non-mechanically coupled and adapted to
communicate with each other independently of controller 1103, including,
for example, feedback and status information of one or more of the
individual components or feedback information. Such coupling of
information or data among individual elements or components of the
pressure/flow loop subsystem 1105 can include pressure/flow loop
component feedback information, such as the status of respective
pressure/flow loop components (see, for example, Ffb in FIG. 27)
and/or feedback data or information, such as FBj or other
information. Such non-mechanical coupling provides a non-mechanical
implementation of mechanical coupling, including but not limited to the
mechanical coupling described in U.S. Pat. No. 7,063,942. Local
processing can also be used, as discussed, for example, with respect to
FIGS. 28 and 27.

[0159]Controller 1103 includes, for example, any control systems discussed
herein including, for example, control systems 70 in FIGS. 1A-3D and
6A-6D. Controller 1103 can receive input information or input data
corresponding to desired dynamic conditions such as desired pressure,
flow and diameter, desired SPA's, sample dimensions and structural
information related to a sample or samples. Controller 1103 can also
receive feedback information such as feedback signals FBj(t)
corresponding to one or more measured dynamic conditions such as
pressure, flow, diameter, velocity, presence, amounts and concentrations
of particles, nano-particles, organic and inorganic molecules and/or any
biological substances, drugs or materials introduced into the fluid in
pressure/flow loop subsystem 1105 or grown or emerging from the specimen
12 and/or the growth of biological materials in specimen unit 10 or as
otherwise discussed herein.

[0160]Input information can also include information regarding the
pathology and degree of pathology to be simulated, the structure and
properties of the sample or samples, the length of time a sample should
be subjected to a particular set of dynamic conditions, the rate and
manner in which the dynamic conditions change or progress over time, the
composition of the fluid and the rate and manner in which the composition
of the fluid changes over time. Controller 1103 can also serve to couple
various types of dynamic conditions such as pressure (P), flow (Q),
diameter (D), length or stretch (L) and twist/torque (T) to shear stress
(WSS), circulation strain (CS), and in turn the SPA, and vice versa as
discussed herein in accordance with preferred embodiments of the
invention.

[0161]Input information can also include information corresponding to
characteristics of signals representing the dynamic conditions including,
for example, the frequency, phase, amplitude, slew rates and/or duty
cycle of the dynamic conditions, which controller 1103 translates into
control signals fj(t) which in turn drive de various components of
the pressure/flow loop subsystem 1105 in accordance with embodiments of
the invention. The dynamic conditions may be characterized by discrete or
continuous random variables or stochastic variables.

[0162]Feedback signals FBj(t) are received by controller 1103, which
correspond to one or more measured dynamic conditions in pressure/flow
loop subsystem 1105, as discussed herein with respect to various
embodiments of the invention. Feedback signals FBj(t) can be dynamic
conditions actually measured at region A of specimen 1112 (as shown in
FIG. 11, in accordance with an embodiment of the invention. Feedback
signal FBj(t) can be measured dynamic conditions at other locations
in pressure/flow loop system 1105, either upstream or downstream from
specimen 12 in pressure/flow loop system 1105. Controller 1103 receives
feedback signals FBj(t) and in turn can produces control signals
fj(t) for pressure/flow loop subsystem 1105.

[0163]FIGS. 17A and 17B show examples of various forms or types of dynamic
conditions g(t). Forms or types of dynamic conditions refers to a
directly or indirectly measurable time varying physical condition of or
related to tubular structures and/or fluids passed therethrough broadly
defined herein. Examples of various forms of types of dynamic conditions
g(t) which can be produced by system 1101 include pressure P(t), flow
Q(t) wall shear stress WSS(t), circumferential strain CS(t,) diameter
D(t), length or stretch (L) and twist/torque (T) as broadly defined
herein in accordance with the embodiments of the invention. System 1101
can simulate one, two, three or more forms or types of dynamic conditions
in states that may occur in biological as well as non-biological systems.

[0164]FIG. 17B lists types that are linked to dynamics of fluid materials.
These include, but are not limited to, for example, concentration of
fluid material (Cfm), expression of fluid material (Efm),
amounts of fluid material (Afm), velocity of fluid material
(Vfm) and flow of fluid material (Qfm).

[0165]As above, region A represents a portion of the specimen 12 or
tubular structure 1112 is said to have dynamic conditions g1,
g2, . . . gn if the measured values of g1, g2, . . .
gn over a region A are substantially within the ratios of

[0166]FIG. 18 shows examples of classes of dynamic conditions that can be
simulated by systems according to various embodiments of the invention.
Classes of dynamic conditions refer to the location of the tubular
structure at which a set of dynamic conditions to be simulated might
occur. Dynamic conditions that occur in vivo are referred to herein from
time to time as in vivo dynamic conditions. In vivo dynamic conditions
include dynamic in vivo bio conditions and hemodynamic conditions.
Dynamic in vivo bio conditions may include, for example, dynamic
conditions that cells, tissues, or organs, experience in vivo other than
hemodynamic conditions. Dynamic conditions can also include
non-biological dynamic conditions found in tubular structures as broadly
defined herein in accordance with alternative embodiments of the
invention.

[0167]FIG. 19 shows a block diagram of controller 1103 which includes a
translator 1113 and a dynamic parameter or dynamic condition generator
1117. Input information can include dynamic conditions represented by
gj(t) according to an embodiment of the invention. For example,
dynamic conditions g1(t), g2(t) and g3(t) could be
pressure P(t), flow Q(t), and diameter D(t), at a region A, respectively.
Input information can be information which is used to characterize the
dynamic conditions gj(t). Input information can be used to retrieve
certain preselected dynamic conditions gj(t) stored in controller 70
and/or generate dynamic conditions and/or associate or link dynamic
conditions or states of physiology as required to produce control signals
for pressure/flow loop subsystem 1105 in accordance with embodiments of
the invention.

[0168]FIG. 20 shows a translator 1113 which receives dynamic conditions
gj(t) and translates those dynamic conditions to N control signals
ft(t) . . . fN(t). The number and characteristics of the
control signals fj(t) depend on the architecture implemented for
pressure/flow loop subsystem 1105 as will be discussed in accordance with
various embodiments of the invention.

[0169]FIG. 21 shows physiological coronary flow Q(t) and pressure P(t) to
be produced by system 1101 at, for example, specimen 12 of FIG. 18. In
this example, the state includes types of dynamic conditions, pressure
P(t), flow Q(t) and diameter D(t) where diameter represents the outer
diameter of a tubular structure. The class of dynamic conditions is in
vivo hemodynamic coronary conditions. A representative signal
corresponding to pressure P(t) can be generated digitally with signal
processing techniques or actually measured by sampling over a period T or
other methods as known to one of ordinary skill in the art. Controller
1103 can perform a Fast Fourier Transform (FFT) on P(t) to yield the
amplitude and phase of P(t) for the first and higher order harmonics. In
embodiments of the invention, amplitude and phase of at least the first
harmonic is determined and/or utilized, and preferably the first two
harmonics, and more preferably the first three harmonics, and more
preferably at least the first 4-10 or more harmonics are determined and
utilized.

[0171]FIG. 22 shows an exemplary pressure/flow loop subsystem 1105 for
system 1101 of FIG. 1B in accordance with an embodiment of the invention.
Pressure flow loop subsystem 1105 includes bellows pumps 405a and 405b
positioned at the upstream and downstream ends 10a and 10b, respectively
of the specimen unit 10 in concert with occluder valves 35-38
respectively positioned upstream and downstream of each of the bellows
pumps 405a, 405b to generate an exemplary dynamic condition A set of
control signals f1-f4 which correspond to the desired condition
are generated by the control system 70 to control the occluder valves
35-38, and dynamic control signals f5 and f6 control operation
of each of the bellows pumps 405a, 405b to generate the desired condition
in the specimen unit 10. For ease of discussion, the valves 35-38 are
either fully open or fully closed. However, it is well understood that
the values 35-38 may at any given time be partially open/closed, and that
appropriate slew rates may be applied to the opening/closing of any of
the valves 35-38 to generate different conditions in the specimen unit 10
as required.

[0172]FIGS. 23a-23d show various stages of bellows pumps 405a and 405b and
FIG. 24 shows states during one cycle, or period T of operation has been
divided into four segments 0-T/4, T/4-T/2, T/2-3T/4, and 3T/4-T. One
period of operation can correspond to cycle of or heart beat or as
described herein in accordance with embodiments of the invention. At time
T=0, as shown in FIG. 23a, the upstream pump 405a is fully expanded and
full of fluid, and the downstream pump 405b is fully contracted, thus
having little to no fluid capacity. Both of the upstream valves 35 and 36
are closed, while the downstream valves 37 and 38 are open, thus
containing the fluid between the valve 35, through the first pump 405a
and the specimen unit, and to the valve 36. As the system moves to the
condition T/4, with the valves 37 and 38 open, the upstream pump 405a
contracts to push the fluid into the specimen unit 10, and the downstream
pump 405b expands to prepare to draw fluid away from the specimen unit 10
and into the pump 405b once the valve 36 is opened. Thus, at time T/4,
the valves 35 and 36 are open, the valves 37 and 38 are closed, the
upstream pump 405a is contracted, and the downstream pump 405b is
expanded.

[0173]As the system moves from this arrangement/condition at T/4, as shown
in FIG. 23b, towards T/2, the valves 35 and 36 open, the valves 37 and 38
close, the upstream pump 405a expands once again fill with fluid, and the
downstream pump 405b contracts to expel fluid from the pump 405b and out
into the downstream end of the flow loop towards the reservoir 20. As the
system moves from this arrangement/condition at T/2, as shown in FIG.
23c, towards 3T/4, the valves 35 and 36 once again close, the valves 37
and 38 once again open, the upstream pump 405a contracts to push fluid
into the specimen unit 10, and the downstream pump 405b expands to draw
fluid from the specimen unit 10 and into the pump 405b.

[0174]From this point, one cycle, or "pulse," is completed as the system
moves to from 3T/4, as shown in FIG. 23d, to time T, where the valves 35
and 36 open, the valves 37 and 38 close, the upstream pump 405a expands
once again fill with fluid, and the downstream pump 405b contracts to
expel fluid from the pump 405b and out into the downstream end of the
flow loop towards the reservoir 20.

[0175]It is noted that, in this particular example, the flow of fluid
through the specimen unit 10 is a substantially regular pulsatile flow in
which fluid is drawn into the specimen unit 10, held there for a given
(small) amount to time, and then drawn out into the flow loop. In this
particular example, simply for ease of discussion, the expansion and
contraction of the bellows pumps 405a, 405b is shown to occur
substantially about the centers of the bellows. However, by expanding
and/or contracting the pumps 405a, 405b in different directions from
those shown in FIGS. 23a-23d, such as by forcing all of the fluid held in
the bellows to flow in a single direction which may be opposite that of
the fluid held in the other bellows, and/or by varying the rate/timing of
the opening and closing of the valves 35-38, numerous different
conditions may be generated. More specifically, as the fluid flows into
and out of the specimen unit 10 through the interaction of the fluid
pushed into and drawn out of the specimen unit 10 by the upstream and
downstream pumps 405a, 405b, numerous different combinations of pressure
and/or flow rate may be generated as the fluid is forced to occupy the
same space and/or change direction as it "collides" in the specimen unit
10, or is simultaneously drawn out of the specimen unit 10a from both the
upstream and downstream ends 10a, 10b.

[0176]An exemplary dynamic condition in which pressure and flow are
substantially in phase, in which SPA is essentially 0°, is shown
in FIG. 25A. In this essentially healthy condition, the valve 35 would
initially be closed and the pump 405a full of fluid which is pumped
through open valve 37 into the specimen unit 10, out of the specimen unit
10 trough open valve 36, where it is stopped by closed valve 38, pushed
back into the specimen unit 10 through the action of the pump 405b, and
then drawn out again through open valves 37 and 38 into the flow loop and
towards the reservoir 20.

[0177]Another exemplary dynamic condition in which pressure and flow are
90° out of phase, or an SPA of essentially 90°
representative of a somewhat diseased condition, is shown in FIG. 25B.
Still another exemplary condition in which pressure and flow are
180° out of phase, or an SPA of essentially 180°
representative of a more severely diseased condition, is shown in FIG.
25C. These conditions may be generated by varying the direction(s) in
which the fluid is moved by the pumps 405a, 405b into and out of the
specimen unit 10, and the varying degrees of pressure and/or flow
disturbance or acceleration experienced as a result.

[0178]FIG. 26 shows a schematic diagram of features of a bellows pump 400
such as bellows pumps 405a and 405b of FIG. 22. Pump 400 is one example
of a pump that can be implemented in systems in accordance with
alternative embodiments of the invention. A first end 406a of bellow 405
is fixed, for example, to a first support 410. The first support 410 is
shown in FIG. 26 as attached to a structure 415 that renders it
substantially unmovable. The second end 406b of the bellow 405 is
attached to a movable support 420.

[0179]The movable support 420 is attached to a movable plate 425, which is
in turn movable by means of a drive system 430 comprising a linear motor
431 and magnetic plate 435 in accordance with an embodiment of the
invention. The linear motor 431 interacts with the magnetic plate 435 to
move the movable plate 425 and therewith the movable support 420 and the
second end 406b of the bellow 405. Other types of drive systems may also
be appropriate.

[0180]The bellow 405 may be made, for example, of plastic, such as
polypropylene, or silicon.

[0181]The drive system 430, and in particular, the linear motor 431 can be
driven by one or more control signals. An encoder unit 440 may be
arranged to include an encoder 440a attached to the movable plate 425,
and a reader 440b, which senses a position of the movable plate 425 and
provides the feedback signal ffb to the pump controller 2701. The
encoder unit 440 may be, for example, a mechanical encoder, an optical
encoder, a capacitive encoder, a magnetic encoder or a laser encoder,
which would include a laser and corresponding reader.

[0182]In this exemplary pump, blood flows into the bellows pump 400 in a
direction of arrow 36 in FIG. 26 via orifice 445 and exits the pump 400
in a direction of arrow A2 via orifice 450. The pump 400 is provided with
the control signal, such as control signal f5(t) discussed above,
received from controller 70, which controls the pumping of the pump 400
to provide the desired flow characteristics. That is, the drive system
430, including linear motor 431 and magnetic plate 435, move the movable
support 420 and the second end 405b of the bellows pump 405 to create the
desired pumping effect in response to the control signal f5. The
feedback signal ffb indicative of the position of the movable plate
425 is provided by the encoder unit 440 to pump controller 2701 to ensure
the desired pumping effect is being created.

[0183]The drive system 430 is driven by a control signal, such as control
signal f5(t) discussed above, received from controller 70. The
control signal f5(t) controls the current to the linear motor 431
via pump controller 2701 shown in FIG. 27, according to an embodiment of
the invention. Pump controller 2701 includes motor controller 2703 and
amplifier 2705. Motor controller 2703 may reside in controller 70. In
alternative embodiments of the invention, motor controller 2703 may
reside in dynamic condition generator 1117 and/or translator 1113. Motor
controller 2703 may independently control one or multiple motors 431.
Feedback signal ffb can be received from encoder 440 by pump
controller 2701 at motor controller 2703 and/or amplifier 2705. An
example motor controller 2703 is SPii Plus HP Series motion controller by
ACS Motion Control. Examples of motors 430 includes AC servo/DC brushless
motors, DC brush motors nanomotion piezo-ceramic motors, step motors and
servo motors. Motor 430 preferably has sub-nanometer resolution such as
those used in semiconductor manufacturing, water inspection, or flat
panel display assembly and testing.

[0184]FIG. 28 shows controller 70 with processor 2711 coupled to pump
controller 2701 and memory 2715 in accordance with an embodiment of the
invention. Motor controller 2703 in pump controller 2701 can be used to
process input information received by controller 70. See also FIGS. 18,
28, 19 and 22. Motor controller 2703 may include a local processor 2709
and memory 2707 such as cache memory or other types of memory. Referring
to FIGS. 19 and 28, the roles of dynamic condition generator 1117 and
translator 1113 can be shared to varying degrees by processor 2711 and
local processor 2709 in motor controller 2703. Pump controller 2701 may
take on the bulk of the processing in controller 70 so that processor
2711 functions merely to synchronize generation of dynamic conditions
gj(t) and translate the dynamic conditions to control signals
fj(t) based on input information to controller 70 according to one
embodiment of the invention. In alternative embodiments, processor 2711
may perform a greater portion of the processing in controller 70. For
example, processor 2711 can generate process input information to link to
pump controller 2701 to yield control signals fj(t). The dynamic
conditions gj(t) can be linked to input information in controller 70
at, for example, local memory 2707 and/or in memory 2715.

[0185]Referring again to FIGS. 18, 19 and 20 in accordance with
embodiments of the invention, control signals fj(t) for pressure
flow loop subsystem 1105 are determined by operating with a first set of
controls signals fj(t) input to pressure flow loop subsystem 1105
and measuring a resulting first set of dynamic conditions and linking
that first set of control signals with the resulting first set of dynamic
conditions. One or more of the control signals are then slightly varied
to yield a second set of control signals, measuring a resulting second
set of dynamic conditions and linking the second set of control signals
to the resulting second set of dynamic conditions. This process is
repeated to form a discrete set of dynamic conditions linked to a
corresponding set of control signals which can be stored, for example, as
a lookup table in controller 70. The number of sets of dynamic conditions
can vary depending on the desired flexibility of system 1101. A variety
of interpolation techniques can also be used to interpolate between sets
of dynamic conditions to provide corresponding sets of control signals
thereby yielding a fully flexible "dial-up" system 1101 capable of
producing sets or states of dynamic conditions between those determined
using the above approach in accordance with yet another embodiment of the
invention.

[0186]FIG. 29 shows steps that may be implemented to develop sets of
control signals corresponding to dynamic conditions and/or input
information according to an embodiment of the invention. Step S1301
involves selection of an initial set of control signals, which can be
written as a vector F1(t) of k control signals, namely,
F1(t)=(f11(t), f12(t) . . . f1k(t)). As an example,
an initial set of control signals can be a sinusoidal signal for pumps,
as described in reference to various embodiments of the invention.
Occluders in dynamic pressure/flow subsystem could be arranged to receive
control signals as shown, for example, in FIGS. 22 and 24. At step S1305,
the initial set of control signals F1(t) are input to the
pressure/flow loop subsystem 1105.

[0187]Step S1306 involves measuring an associated or corresponding set or
state of dynamic conditions, which can be written as a vector G1(t)
of M dynamic conditions, namely, G1(t)=(g11(t),g12(t) . .
. g1M(t)). Step S1307 involves linking or associating the resulting
dynamic conditions g1j(t) to the initial stage or set of
control signals f1j(t). Linking can include storing a lookup
table in memories 2701 and/or 2705 (see FIGS. 28 and 27) of controller 70
or 1103 according to an embodiment of the invention. Step S1307 may also
include storing characteristics of the measured dynamic conditions
g1j(t) associating pathological as well as the shape or
characterization of the signals representing the dynamic conditions to
the control signals. Step S1309 involves adjusting or perturbing one or
more control signals f1j(t) to yield a second set or state of
control signals F2, which include element control signals
f2j(t), then measuring the resulting second set or state of
dynamic conditions G2, which include element dynamic conditions
g2i(t).

[0188]This process can be repeated to produce a desired number of links
between input information and/or dynamic conditions Gm and control
signals Fm in accordance with embodiments of the invention. For
example, implementation of steps S1301-S1309 involves assigning pumps in
pressure/flow loop subsystem 1105, a sinusoidally varying control signal
corresponding to position of the bellows versus time (as discussed with
respect to bellows pumps 400 in FIG. 26) with a frequency approximately
equal to a base heart rate. Step 1309 may then involve varying the phase
of one of the bellows pumps with respect to the other bellows pump to
establish a next set of control signals Fm and measuring the
resulting set of dynamic conditions Gm.

[0189]Alternatively, step 1309 might involve varying the amplitude or
stroke length of one of the bellows pumps with respect to the other to
establish a next set of control signals Fm. Also, in accordance with
linear and non-linear interpolation techniques, sets or states of dynamic
conditions can be linked to associated sets of control signals to enable
"dial-up" dynamic conditions.

[0190]Controller 70, 1103 can further be trained to produce dynamic
conditions that evolve over time. This includes adjusting the frequency,
phase or amplitude of the first and/or higher order harmonics of one or
more dynamic conditions over multiple periods T of pulses.

[0191]For example, the first order frequency ω1 of one or more
types of dynamic conditions can vary over time TTi in a
predetermined manner. For example, the resting heart rate might be at 70
beats per minute. The dynamic conditions such as pressure P(t), flow Q(t)
and/or diameter D(t) could gradually change from a first order frequency
of 70 Hz to 130 Hz over a time span of minutes, hours, etc. The rate and
progression of change for different order harmonics may differ for any
one dynamic condition as well as for different types of dynamic
conditions P(t) versus Q(t), D(t), etc. This holds for the phase
θj of the first or higher order harmonics of one or more
dynamic conditions.

[0192]FIG. 30 shows variations over time in the first order harmonic
ω1(t) of a dynamic variable g(t) which can be produced in
accordance with an embodiment of the invention. At t=0, ω1(t)
might correspond to a resting heart rate ω1(0)=1/T (say 70
cycles per second), where T is the period of the heartbeat. The first
order frequency ω1(t) then increases to ω1(t1) (say
120 cycles per second) over multiple periods T (e.g. 5T, 10T, 100T, 1000T
. . . ) at time t1 (t1=5T, 10T, 100T, 1000T . . . ). Between
time t1 and t2, the first order harmonic ω1(t)
remains relatively constant at ω1(t2) then decreases to
ω1(t)=ω1(t3) (say 100 cycles per second) at time
t3. Between time t3 and t4, ω1(t) again remains
relatively constant at ω1(t3) and then increases back to
ω1(t1) at t5 and continues to increase for t>t5.
The above holds for higher order frequencies ω1j(t) as well in
accordance with embodiments of the invention.

[0193]FIG. 31A shows an example of the variations in time of the phases
θj of the first three harmonics for j=1, 2 and 3 of a dynamic
condition g(t). The corresponding first three harmonics ω1(t),
ω2(t) and ω3(t) could remain constant or themselves
varied over time in accordance with alternative embodiments of the
invention. The number of harmonics whose phase and amplitude are utilized
can be preferably 1, and more preferably at least 2 and more preferably
at least 3 and more preferably at least 4-10. The same holds for multiple
forms or types of dynamic conditions it being understood that the
frequency ω1(t) and phase θj(t) of one dynamic
condition g1(t) may differ from the frequency ω1(t) and
phase θj(t) of a second or additional dynamic variables
g1(t) in accordance with embodiments of the invention. In these
embodiments of the invention, system 110 can be used to simulate a person
or mammal exercising or exerting effort in any physical activity,
experiencing shock, disease or any other situations which could naturally
occur.

[0194]FIG. 32 shows variations of the jth harmonic amplitude Gj
of a dynamic condition g(t) where

g ( t ) = j = 1 N G j μ j (
ω j t + θ j ) ( 1 )

where μj(ωjt+θj) are normalized basis
functions of dynamic condition g(t), such as sinusoids. Just as
ωj(t) and θj(t) may vary over time T, the amplitude
Gj can vary over time in accordance with embodiments of the
invention.

[0195]FIG. 32 presents one example of how the amplitude of the
jth harmonic of dynamic condition g(t) (initially Gj(0) at
t=0), increases to Gj(t1) at t=t1. The amplitude
Gj(t) remains at Gj(t1) until t=t2, then increases to
Gj(t3) at t=t3, where it remains until t=t4 at which
point it decreases to Gj(t1) at t=t5. Dynamic condition
g(t) may be one of the types (FIGS. 17A and 17B) of dynamic conditions
from one or more states or classes (FIG. 18) of dynamic conditions.

[0196]FIG. 31B shows an example of how amplitudes of the first three
harmonics of a dynamic condition may vary with time as well as the
corresponding dynamic condition in real time. At t=0, the first order
amplitude G1(0)>G2(0), the second order amplitude G2(0)
is about 0.8 G1(0), and the third order G3(0) is about half of
G1(0). The presence of the higher order terms for dynamic variable
G(t) are apparent as variations in the plot of G(t) versus time. As t
approaches t1, the second and third order amplitudes G2(t) and
G3(t) approach zero, which results in the dynamic condition G(t)
varying more sinusoidally.

[0198]System 1101 produces an experience G at region A in a tubular
structure in accordance with embodiments of the invention. An experience
can correspond to actual dynamic conditions experienced at region A of
tubular structures in vivo or actual dynamic conditions experienced at
region A of tubular structures (including non-biological dynamic
conditions) that are not in vivo. In addition, an experience can
correspond to dynamic conditions which are used to train or condition a
tubular structure.

[0199]For example, three experiences GA(t), GB(t) and GC(t)
can be represented as:

GA=(g1A(t),g2A(t) . . . gNA(t))

GB(t)=(g1B(t),g2B(t) . . . gNB(t) . .
. gN'B(t))

GC(t)=(g1C(t),g2C(t) . . . gNC(t) . .
. gN'C(t) . . . gN''C(t))

where the experiences may be actual in vivo, actual non-biological,
training or conditioning and/or combinations thereof in accordance with
embodiments of the invention. The types of dynamic conditions FIGS. 17A
and 17B) for experiences GA, GB or GC are not necessarily
the same. For example, g1A is not necessarily the same as
g1B(t) or g1C(t). Also, the number of dynamic
conditions N, N' or N'' can be different in accordance with embodiments
of the invention.

[0200]FIGS. 33A and 33B show representative frequencies ωij(t)
and amplitudes Gij(t) for three different physiological experiences
i=A, B, and C, respectively. Training time corresponds to the length of
time that a set of dynamic conditions are to be produced at a tubular
structure before they are repeated. Total training time TTTi
corresponds to the total time a tubular structure is subjected to a set
of dynamic conditions for the ith physiological experience.

[0201]FIG. 34 shows an example of how systems such as systems 1 and 1101
produce a single experience using three dynamic variables P'(t), Q'(t)
and D'(t) and which exhibit a pattern of variations over a training time
TTi which is repeated for a total training time TTTi=4
TTi. Hence, P', D' and Q' can represent, for example, the amplitude,
phase or frequency of the pressure, flow and diameter at a specimen or
tubular structure 12, 1112. In accordance with embodiments of the
invention, total training time TTTi can be multiple training times
TTi and can include fractions of training time TTTi. For
example, TTTi=xTTi, where x is a real number, for example
x=0.3, 1 5/3, 20, 100, 1000 . . . and so forth.

[0202]FIG. 35A shows how systems 1, 1101 can be used to produce dynamic
conditions which would be experienced by in vivo tubular structures in a
patient with a particular patient history while undergoing a
physiological experience. In particular, the dynamic conditions are
reproduced at a specimen or tubular structure 12 or 1112 inserted into
systems such as systems 1, 1101 in accordance with embodiments of the
invention.

[0203]Step 3501 involves inputting physical characteristics of an in vivo
tubular structure located in a patient and inputting patient history
information. FIG. 35B shows exemplary patient history information.

[0204]Step 3503 involves either selecting a non in vivo tubular
corresponding to the in vivo structure, or removing the in vivo tubular
structure from the patient. Step 3505 involves inserting the selected
tubular structure into system 1, 1101. Step 3507 involves selecting a
physiological experience, examples of which are shown in FIG. 35B.

[0205]Step 3509 involves implementing the selected physiological
experience using system 1, 1101 with the selected tubular structure and
optionally adding, subtracting and/or altering the fluid and/or fluid
material in the system 1, 1101 for a time TTTi. Step 3511 involves
testing, removing and/or outputting resulting fluid from the flow loop of
system 1, 1101 and/or testing removing and/or outputting the resulting
tubular structure from the system 1, 1101.

[0206]In accordance with additional embodiments of the invention, it
should be understood that the number and types of dynamic variables used
during training time TTi for a particular physiological experience
can vary during a training time TTi. For example, system 1101 could
produce 2 dynamic variables g1(t) and g2(t) while measuring or
monitoring the resulting third dynamic variable g3(t), then produce
dynamic variables g2(t) and g3(t) while measuring or monitoring
the resulting first dynamic variable g1(t). The measured/monitored
dynamic variable can serve as feedback signals FB1 (see, for
example, FIG. 18) for controller 1103 in accordance with various
embodiments of the invention.

[0207]The physiological experiences can be selected to train a tubular
structure (biological or non biological as discussed above) and are
represented by two or more types of dynamic conditions (or variables) for
any state or class of dynamic conditions. For example, physiological
experience A might have a training time TTA=24 hours with dynamic
conditions GA(t) made up of a set of the three dynamic conditions
pressure P'(t), diameter D'(t) as broadly defined herein, and flow Q'(t)
experienced by a tubular structure located in the pulmonary artery of a
60-year old athletic male Caucasian (patient history) reported over
multiple training times TTA. Physiological experience B might, for
example, correspond to a training time of TTB=1 week with dynamic
conditions GB(t) made up of a set of two dynamic conditions
experienced by a tubular structure located in the large intestine of a
25-year old athletic paraplegic (patient history). Again, other examples
of physiological experiences are listed in FIG. 35B, it being understood
that system 1101 is not necessarily limited physiological experiences
listed therein.

[0208]Controller 1101 can be characterized by its flexibility, the classes
(FIG. 18) of dynamic conditions and/or types (FIGS. 17A and 17B) of
dynamic conditions that can be produced at a given region of a tubular
structure.

[0209]Controller-Flexibility

[0210]For a given pressure/flow loop subsystem 1105, controller 1103 may
be trained to provide a single state of dynamic conditions, (a single
state controller) in accordance with embodiments of the invention.
Similarly, for a given same pressure/flow loop subsystem 1105, controller
1103 may be trained to provide discrete states or sets of dynamic
conditions, (a discrete controller) in accordance with other embodiments
of the invention. Also, for a given same pressure/flow loop subsystem
1105, controller 1103 may be trained to provide multiple discrete and
continuous states of dynamic conditions (a hybrid controller) in
accordance with embodiments of the invention. Similarly, for a given
pressure/flow loop subsystem 1105, controller 1103 may be trained to
provide a single physiological experience (FIG. 34) (a single experience
controller), multiple physiological experiences (a multi-experience
controller), and a hybrid of discrete physiological experiences and also
the flexibility to dialup various states of dynamic conditions, (a hybrid
experience controller).

Controller--Type or Form (FIGS. 17A and 17B) of Dynamic Conditions

[0211]For a given pressure/flow loop subsystem 1105, controller 1103 can
be trained to output control signals fj(t) which yield certain types
of forms FIGS. 17A and 17B) of dynamic conditions. For example,
controller 1103, can be trained to output control signals that produce
g1(t), g2(t), g3(t) and g4(t) at a region A of a
tubular structure, where g1(t) is pressure P(t), g1(t) is flow
Q(t), g3(t) is the wall thickness along a first direction and
g4(t) is the circumferential strain at region A.

Controller--Class of (FIG. 18) of Dynamic Conditions

[0212]For a given pressure/flow loop subsystem 1105, controller 1103 can
be trained to output control signals fj(t) which yield states of a
particular class (FIG. 18) of dynamic conditions. For example, if
controller 1103 is trained to output control signals fj(t) to a
given pressure/flow loop subsystem 1105 which yield one or more states in
the dynamic bio class of conditions for example, controller 1103 is a
non-invivo condition controller, and system 1101 is a dynamic bio
condition system.

[0213]For a given pressure/flow loop subsystem 1105, system 1101 may be
referred to as a single state system if controller 1103 is a single state
controller, a discrete state system if controller is a discrete state
controller, a hybrid system if controller is a hybrid controller, and a
dial-up system if controller is a dial-up controller in accordance with
embodiments of the invention.

[0214]Similarly, for a given pressure/flow subsystem 1105, system 1101 may
be a single experience system if controller 1103 is a single experience
controller, a multi-experience system if controller 1103 is a
multi-experience controller, and a hybrid experience system, if
controller 1103 is a hybrid experience controller.

[0215]The method and systems described herein can be characterized by
additional/alternative means in accordance with embodiments of the
invention as shown in FIGS. 36, 37, 39 and 38. FIGS. 36 and 37 show block
diagrams of system 1101 with controller 1103 and a pressure/flow
subsystem 1105 which together generate a flow loop 2105 of fluid as
broadly defined herein. System 1101 of FIG. 36 includes a specimen unit
10 in accordance with embodiments of the invention, whereas system 1101
in FIG. 37 shows a block diagram with controller 1103 and a pressure/flow
subsystem 1105 with a flow loop 2105 of fluid, but without a specific
specimen unit 10. Instead, a region of the flow loop 2105 itself serves
as the region A of a tubular structure (e.g., FIG. 11) in accordance with
embodiments of the invention. Controller 1103 is trained to generate
control signals fj(t) which when input to pressure flow subsystem
1105 produce various dynamic conditions and/or physiological experiences
at a tubular structure in accordance with embodiments of the invention.

[0216]FIGS. 38 and 39 show system 1401 with pressure/flow loop subsystems
1105 in accordance with embodiments of the invention. Here pressure/flow
loop subsystem 1105 does not include flow loop fluid but does include a
conduit 3701, which operatively couples pressure/flow loop subsystem
components, in accordance with various embodiments of the invention.
Conduit 3701 can include any tubes, pipes, cylinders, tubular structures
and any other coupling components described herein with respect to
systems such as systems 1 and 1101 and others known to those of ordinary
skill in the art. As with system 1101 of FIG. 36, system 1101 of FIG. 39
includes a specimen unit 10 in accordance with an embodiment of the
invention. Similarly, as with system 1101 of FIG. 37, system 1101 of FIG.
38 does not include a specimen unit 10. Instead, a region of the
pressure/flow loop subsystem 1105 itself serves as the region A of the
tubular structure (FIG. 11) in accordance with embodiments of the
invention. Again, controller 1103 is trained to generate control signals
fj(t) which when input to pressure/flow loop subsystems 1105 produce
various dynamic conditions at a region A in pressure/flow loop subsystem
1105.

[0217]FIG. 40 shows system 1101 with sensors 1, 2n, A and Bn. Although
four sensors are shown as an example, purposes, number and types can be
greater or smaller than four. Sensors 1) 2n, A and/or Bn include, for
example transmitters, receivers, transmitter/receivers, transducers,
detectors and other sensors. Fluid sensors as used herein are sensors in
the fluid which are added to the pressure/flow loop subsystem of FIGS. 38
and 39 or which comprise the flow loop shown in FIGS. 36 and 37.

[0218]System sensors (A) include any transmitters, receivers,
transmitters/receivers, transceivers, transducers, detectors, as well as
any devices which can be used to detect images or measure any one or more
parameters related directly or indirectly to one or more dynamic
conditions, such as those listed in FIGS. 17A and 17B and discussed
herein.

[0219]FIGS. 41A-44C show three electrode configurations for measuring the
conductivity of a fluid and/or a monolayer in a tubular structure 1112,
in accordance with embodiments of the invention. In the embodiment of
FIG. 17C, electrodes 1200 and 1202 are placed on opposite sides of the
tubular structure 1112 and are connected to a voltage source 1204. In the
embodiment of FIG. 17D, ring electrodes 1206 and 1208 are spaced apart
and extend around at least a portion of the circumference of the tubular
structure 1112, and preferably around the entire circumference of the
tubular structure 1112. In the embodiment of FIG. 17E, electrodes 1200
and 1202 are placed on one side of the tubular structure 1112.

[0220]The three electrode configurations of FIGS. 41A-44C measure the
conductivity of the fluid inside the tubular structure 1112 and/or a
monolayer inside the tubular structure 1112 along different directions.
For example, the configuration shown in FIG. 17E is particularly useful
for measuring the conductivity of a monolayer (not shown) grown on the
inside surface of the tubular structure 1112. Such a conductivity reading
could be used, for example, to measure the functionality and/or the
integrity of the monolayer in the tubular structure 1112.

[0221]The voltage source 1204 can be a direct current source or an
alternating current source. Thus, the term "conductivity", as used
herein, includes the measurement of resistivity, impedance and reactance.

[0223]System nanosensors (Bn) represent nanosensors, nanotransmitters,
nanoreceivers, nanotransceivers, nanotransducers, nanodetectors, as well
as any devices which can be used to detect, image or measure one or more
parameters related directly or indirectly to one or more dynamic
conditions, including, but not limited to, those listed in FIGS. 17A and
17B.

[0224]Referring back to FIG. 40, fluid sensors 1 include any transmitters,
receivers, transmitters/receivers, transceivers, transducers, detectors,
as well as any devices which can be used to detect images or measure any
one or more parameters related directly or indirectly to one or more of
the dynamic conditions, including those listed in FIGS. 17A and 17B.

[0225]Fluid sensors 2n represent nanosensors, nanotransmnitters,
nanoreceivers, nanotransceivers, nanotransducers, nanodetectors, as well
as any devices which can be used to detect, image or measure one or more
parameters related directly or indirectly to one or more dynamic
conditions, including, but not limited to, those listed in FIGS. 17A and
17B.

[0226]FIG. 42A shows examples of how exemplary sensors A, Bn, 1 and 2n can
be communicatively coupled or can transmit, receive, transmit and
receive, detect and forward data related to, for example, dynamic
conditions and/or other data used as feedback FBj(t). Arrows
indicate direction of flow of data or information. Dashed lines are used
to indicate all possible data flow it being understood that actual
information flow depends on the type of sensors. In some embodiments of
the invention, sensors may not directly measure, but may instead serve as
boosters or repeaters. For example, in one embodiment of the invention
the fluid contains thousands of nanodetectors and receivers which detect
one or more dynamic conditions and transmit photons of a certain
frequency depending on the presence of certain gases, liquids, solids
and/or biological materials, which in turn can be detected by system
sensor A (e.g., photon detector), which in turn puts out a feedback
signal FBj(t) to controller 70. Referring to FIG. 42A, this would be
represented by the configuration shown in FIG. 42B.

[0227]FIG. 43A shows various possibilities of how six sensors A, Bn, C, 1,
2n and 3n can be communicatively coupled or transmit, receive, transmit
and receive, detect and forward data related to, for example, dynamic
conditions or other data used as feedback FBj(t). Again, in some
embodiments of the invention, sensors may not directly measure, but may
instead serve as boosters or repeaters. Referring to FIG. 43A, this would
be represented by the configuration shown in FIG. 43B.

[0228]According to embodiments of the invention, a receiver (e.g., system
or fluid) can have exemplary volumes of less than 1 cm2, less than
500 mm2, less than 1 mm2, less than 500 μm2, less than
100 μm2, less than 1 μm2, less than 500 nm2, less
than 100 nm2, less than 1 nm2 or the like. According to other
embodiments of the invention, at least one receiver (e.g., system or
fluid) can have exemplary dimensions of less than or at least 500 mm
along a first direction, 1 mm along a first direction, 500 μm along a
first direction, 100 μm along a first direction, 1 μm along a first
direction, 500 nm along a first direction, 100 nm along a first
direction, 50 nm along a first direction, 1 nm along a first direction,
0.1 nm along a first direction or the like. In other embodiments, a
receiver can have a similar size to a fluid receiver. In other
embodiments, a transmitter, fluid transmitter, a transmitter/receiver,
fluid transmitter/receiver can have a similar size to a fluid receiver or
receiver. In other embodiments, a sensor (e.g., in a system, fluid,
specimen or the like) can have similar sizes to a receiver, transmitter,
or transmitter/receiver.

[0229]Fluid sensors, system sensors and/or specimen sensors (A, B, . . .
An, Bn, . . . 1, 2, . . . 1n, 2n, . . . ) discussed
herein (e.g., receivers, transmitters, transceivers) may further include
probes in accordance with embodiments of the invention. Probes can be
used as fluid, system and/or specimen sensors. Probes can characterize
biological activity such as cell activity. For example, biological
activity can be observed or detected using activity based probes. An
activity based probe can form a bond (e.g., irreversible covalent bond)
with a desired active biological target (e.g., protein target). Once the
target is coupled to the probe, the target can be more easily detected,
monitored or utilized.

[0230]FIG. 44A shows an activity based probe D01 can include an engaging
end D05 or warhead, a tag D09 and a connector portion D11 in accordance
with one embodiment of the invention. Engaging end D05 can be designed to
engage amino acid residue at an active enzyme site D13. Accordingly, the
reactivity, polarity, charge, size and structure can set the
effectiveness and selectivity of the probe. Tag D09 is used to detect
and/or enrich the active target. Tag D09 can include a radioactive
molecule, fluorescent or the like. Tag D09 can be, for example, a biotin,
a radioactive molecule, or a fluorescent molecule such as fluorophore or
125I. Connector portion D11 is a molecular chain which can reduce
interference between tag D09 and engaging end D05 as well as to assist in
selecting the probes target D13 can be, for example, a peptide, alkyl
polyether or the like. Engaging end D05 can be a phosphonate,
fluorophosphonate, epoxyketone or the like.

[0231]FIG. 44B shows a probe D51 which is smaller and preferably
significant smaller than target D53. Here, probe D51 also has an engaging
end D05 and a connector portion D11 but with tag D09 replaced with tag
D55 which includes a drug or pharmacological agent or any small or large
molecules (for example, see FIG. 17B). Hence probe D51 can provide a
delivery mechanism such as a drug delivery mechanism to tubular
structures in accordance with embodiments of the invention. In an
alternative embodiment of the invention, tag D55 can be a combination of
tag D09 of FIG. DA and a drug, pharmacological agent or any other small
or large molecules, in which case probe D51 can serve both as a delivery
mechanism and a sensor for the tubular structure or specimen and/or to
fluid materials in fluids described herein and/or to flow loop fluids in
systems such as systems 1, 1101 in accordance with other embodiments of
the invention.

[0232]FIG. 44B shows yet another embodiment of probe D51 (dashed lines) in
which a second tag D57 is attached with a second connector portion D61 in
accordance with another embodiment of the invention. Hence, probe 51 with
tag D55 functions in a similar manner to that described above with
respect to FIG. DA, and probe 51 with tag D57 functions as a delivery
mechanism as described herein.

[0233]FIG. 45A shows tubular structures 1112 which are permeable or
semipermeable to fluid materials of any kind, and shown as permeable
tubular structures 1152, in accordance with embodiments of the invention.
Permeable tubular structures 1152 allow for the migration, flow and/or
diffusion of fluid and/or any fluid materials, such as particles,
sensors, or molecules as described herein, examples of which are listed
in FIGS. 17A and 17B. Hence, measurement of the amount, flow, velocity of
fluid or fluid material corresponds to measurement of types of dynamic
conditions, examples of which are shown in FIGS. 17A and 17B.

[0234]The direction of velocity and flow can include measurement of a
directional dynamic condition g(t) having a component in the vertical
direction, as well as measurement of nondirectional dynamic conditions
g(t), such as amounts of fluid or fluid material. System and/or fluid
sensors can be used to measure these types of dynamic conditions, in
accordance with embodiments of the invention.

[0235]FIG. 45B shows other types of tubular structures 1112, which are
porous or semi porous tubular structures 1155, in accordance with
embodiments of the invention. Again, tubular structures 1155 allow for
the migration, flow and/or outfusion of fluid and/or fluid material as
described herein, examples of which are shown in FIGS. 17A and 17B.
Hence, measurement of the amount, flow, velocity or other dynamic
condition of fluid or fluid material constitutes measurement of types of
directional dynamic conditions g(t) and/or measurement of types
non-directional dynamic conditions g(t), as shown in FIGS. 17A and 17B.
Again, directional dynamic variables may include a nonzero component in
the radial direction.

[0236]FIG. 45C shows other types of tubular structures 1112 which are
electrospun tubular structures 1157, preferably made of fibrin in a
manner such as that described, for example, in U.S. Pat. Nos. 6,592,623
and 6,787,357, the contents of which are incorporated herein by
reference. Electrospun tubular structures 1157 can be permeable and/or
porous.

[0237]FIG. 46 shows other types of tubular structures 1112, which are
microgrooved tubular structures 1252. Microgrooved tubular structures
1252 have depths of 50 nm and 700 nm, can have grooves with widths of
between 40 nm and 2000 nm and preferably 70 nm and 1400 nm, and pitch
between approximately 200 to approximately 5000, and preferably
approximately 400 to approximately 4000 depending on the class of dynamic
condition (FIG. 18) which it will be subjected to, as well as the type of
cells and/or coatings which might be applied to it.

[0238]Tubular structures 1112 can be combinations of two or more of the
above tubular structures, such as two or more tubular structures 1112 in
FIGS. 11, 12, 13A, 13B, 14, 14, 16, tubular structures 1152, 1155, 1157
and 1252 and specimens 12 in FIGS. 1A, 2A-2E, 3A-3D, 5A-5D and 6A-6E.
Hence, a tubular structure could be a combination of a porous tubular
structure 1155 (FIG. 45B) and a microgrooved tubular structure 1252 (FIG.
46).

[0239]Second order dynamic conditions can include any type of dynamic
conditions such as those listed in FIGS. 17A and 17B and any others known
in the art. Second order dynamic conditions are localized dynamic
conditions produced by systems discussed herein, such as systems 1 and
1101, in conjunction with tubular structures having perturbed physical
characteristics and/or properties when those systems operate to provide a
given set of global dynamic conditions and/or dynamic conditions at one
or multiple regions A in accordance with embodiments of the invention.
When these systems operate to provide a given set of global dynamic
conditions and/or known types of dynamic conditions at one or multiple
regions A in accordance with embodiments of the invention.

[0240]Such perturbed tubular structure characteristics include, for
example, shape, structure, porosity, permeability, dimensions, thickness,
stiffness, elasticity, ridges, localized stiffness and elasticity,
protrusions, bumps, rigid full rings or rigid partial rings, expandable
full rings or partial rings with known elasticities, as well as rigid
full sleeves or rigid partial sleeves, or full or partially expandable
sleeves with known elasticity. Other perturbed tubular structure
characteristics can include coatings with altered coefficients of
friction, smoothness and/or roughness of the inner surface of the tubular
structure, and the spatial frequencies of any repeating structure
perturbation, such as small bumps, rings, grooves, sleeves, shapes and so
forth, examples of which will be discussed with respect to FIGS. 47A-47H.

[0241]These dynamic conditions are referred to herein as second order
dynamic conditions because they result the use of the perturbed tubular
structures in systems which operate to provide predetermined or known
global dynamic conditions of one or multiple regions A. Second order
dynamic conditions can be used, for example, to effect additional sets of
dynamic conditions which might be present in vivo for healthy, diseased,
or other in vivo tubular structures. Second order dynamic conditions can
also be used, for example, to create additional sets of dynamic
conditions or other non-biological situations, as well as dynamic
conditions useful for training and testing, or growing tubular
structures, samples of which are shown in FIG. 18.

[0242]All tubular structures discussed throughout can include biological
material, such as cells, etc., can include a hybrid of biological
material and non-biological material, synthetic or non-synthetic
non-biological material or completely biological material, such as veins
or arteries or tissues, or organs and so forth as described herein.

[0243]Variations in cross-sectional area along the z direction correspond
to variations in D as broadly defined herein. Hence, tubular structure
along Z can be represented by D(z). The z axis can represent an
approximately straight line along the direction of the mean pulsatory
flow in tubular structures, in accordance with embodiments of the
invention. Alternatively, the Z direction can represent a line fiat
follows approximately along the center of each cross-sectional area of a
tubular structure, according to other embodiments of the invention.

[0244]Tubular structures also include, for example, biological or
non-biological or hybrid biological and non-biological tubular structures
which have been in any way slightly, moderately or substantially modified
as a result of being subjected to one or more sets of dynamic conditions
and second order dynamic conditions for an amount of time sufficient to
yield any such slight, moderate or substantial modifications of the
tubular structure itself. Again, tubular structures can have perturbed
physical characteristics and/or properties which immediately yield
desired dynamic conditions, including second order dynamic conditions,
once placed in systems described herein with the appropriate global
dynamic conditions, including systems 1 and 1101, according to
embodiments of the invention.

[0245]FIGS. 47A-47H show examples of tubular structures or specimens 12,
1112 which can be used to effect second order dynamic conditions. Again,
as discussed herein, these tubular structures and specimens, as well as
all other specimens and tubular structures including, for example, anyone
or more combinations of those shown in FIGS. 1A, 11, 12, 13A, 13B, 14,
15, 16, 36, 38, 42, as well as any portion or section of systems, such as
systems 1, 1101 which can pass fluid from one location to another as
defined herein (see FIGS. 11 and 12), can be porous, non-porous,
permeable or non-permeable or any hybrid thereof biological,
non-biological or any hybrid thereof, multilayered, multi-channeled or
multiple branched and any combination of these and one or multiple
tubular structures shown in FIGS. XA-XH. Again, tubular structures can
have perturbed physical characteristics and/or properties which nearly
immediately yield the desired dynamic conditions including second order
dynamic conditions once placed in systems 1, 101 and/or 1101 with the
appropriate global dynamic conditions.

[0246]FIG. 47A shows a tubular structure 12, 1112 with varying diameter D
along a z direction which, in accordance with embodiments of the
invention, corresponds to variation of the cross-sectional areas of
tubular structures along the z direction. Again, diameter D, and hence at
cross-sectional areas at Z1 . . . Zn of tubular structures as
defined herein can include FIGS. 12, 13A, 13B and 17. Hence, D1 may
correspond to a circular cross-sectional area and D2 may correspond
to an ovular cross-sectional area. Generally, D1 and D2 can be,
for example, any cross-sectional area including those, for example, in
FIG. 12, and the transition from D1 to D2 along the z direction
can be any series of cross-sectional areas. FIGS. 47B, 47C, 47D show more
examples of tubular structures in which D1 and D2 might be
approximately the same, but the transition of cross-sectional areas
varies along the z direction by becoming larger then smaller (FIG. 47B)
or becoming smaller then larger FIG. 47C).

[0247]FIG. 47D shows another example of tubular structures with
cross-sectional variations along the z direction. If z represents the
approximate center of cross-sectional areas D(z), z=z1-zn, then
the first cross-sectional area D(z1) might represent a circle having
a radius of r1. D(z2) might represent a cross-sectional area
which is ovular on the top half with a major axis radius r2, and
circular on the bottom half still with the radius r1, where
r2>r1. Cross-sectional area D(z3) might be ovular with
a major axis of r3 and a minor axis r4, where
r3>r4 and, for example, r4>r1.

[0248]FIG. 47E shows a tubular structure with grooves 2500 according to
additional embodiments of the invention. FIG. 47E has grooves angled with
respect to the Z direction. Here, the grooves are considered completely
aligned with the z direction if they run approximately parallel to the z
direction. It should be understood that grooves can be any grooves,
microgrooves, ridges, indentations and can be on the inner diameter, the
outer diameter (to effect, for example, a particular flexibility of
elasticity) of the tubular structure and/or within the wall of one or
more layers of the tubular structure, according to embodiments of the
invention. Grooves, as used herein, include the presence and/or absence
of any biological and/or non-biological materials including, but not
limited to, materials used or present in any tubular structures as
defined herein. Accordingly, grooves can be troughs having a desired
cross-sectional shape such as a "V" shape, semi or partially circular or
ovular shape, rectangular shape and so forth. Variations in the depth,
width, length, direction, shape and/or periodicity (for example, distance
between grooves) can produce or alter the resulting second order dynamic
conditions for a given set of global dynamic conditions and/or dynamic
conditions at region(s) A.

[0249]FIG. 47F shows a tubular structure with projections on an interior
surface according to additional embodiments of the invention. Bumps 2515
can provide fluid perturbations as desired or that provide selected
empirical results. Bumps 2515, as used herein, include the presence
and/or absence of any biological and/or non-biological materials
including, but not limited to, materials used or present in any tubular
structures as defined herein. Bumps can have a desired cross-sectional
shape such as a circular shape, semi or partially circular or ovular
shape, rectangular shape and so forth. Variations in the depth, width,
length, direction, shape and/or periodicity (for example, distance
between bumps) can produce or alter the resulting second order dynamic
conditions for a given set of global dynamic conditions and/or dynamic
conditions at region(s) A.

[0250]FIG. 47G shows a tubular structure 12, 1112 in accordance with
another embodiment of the invention. Tubular structure 12, 1112 has a
partial ring 4001, a full ring 4003, a full sleeve 4005, a partial sleeve
4007 and a patch 4009 attached and/or coupled to tubular structure 12,
1112 and/or fabricated into tubular structure 12, 1117. Rings 4001 and
4003, sleeves 4005 and 4007 and patch 4009 can be ridged or flexible to
provide known variations in flexibility and elasticity of the walls of
tubular structure 12, 1112 along the z axis. Such known variations in
flexibility and elasticity yield sets of dynamic conditions including
second order dynamic conditions at tubular structure 12, 1112 which
correspond to certain desired dynamic conditions when used in systems
herein in accordance with embodiments of the invention such as systems 1,
1101.

[0251]As discussed above, FIGS. 47A-47G are tubular structures which can
be formed outside systems 1, 1101 or tubular structures that are formed,
trained and/or grown after being subjected to predetermined dynamic
conditions, including global and/or dynamic conditions at regions A for a
predetermined amount of time. Hence, an initial tubular structure with
initial perturbations and predetermined variations in shape D(z), can
develop a desired shape and desired perturbations to effect the desired
second order dynamic conditions as a result of the growth and/or further
development of grooves, varying elasticity and/or any other perturbations
of the tubular structure due to the growth and/or training of biological
material on and/or the tubular structure.

[0252]Also, tubular structures can be a combination of one or more tubular
structures with biological or non-biological materials which can be
applied to the inner surface and/or the outer surface of the tubular
structures. In addition, tubular structures include all of the above
combinations which have been placed in the systems described herein and
allowed to develop, grow under desired dynamic conditions for a desired
length of time. Such tubular structures are said to have been tubular
structures as shown in FIG. 18 under training/testing dynamic conditions
and, in particular, non-in vivo bio training/testing conditions. In vivo
dynamic conditions might also serve as training/testing conditions as
well as combinations of in vivo dynamic conditions and training/testing
dynamic conditions.

[0253]Tubular structures can be slightly, partially, substantially or
completely trained in the absence of any biological materials as well.
This might include subjecting tubular structures to training by the
systems, in accordance with other embodiments of the invention in order
to prepare them or alter them or test them for a particular use.

[0256]Systems herein including systems 1 and 1101 can be used to model or
simulate. According to one embodiment, systems 1 and 1101 together with
perturbed tubular structures can model pathology or the departure or
deviation from a normal condition at the tubular structure. This can
include anatomic or functional manifestations of a disease (or structural
and functional changes in cells, tissues and organs that underlie
disease). Systems 1 and 1101 can model pathologies within various classes
of dynamic conditions (e.g., see FIG. 18) using at least one and
typically multiple types of dynamic conditions (e.g., see FIGS. 17A and
17B) according to embodiments of the invention. Accordingly, embodiments
of systems 1 and 1101 can be used to determine or evaluate dynamic,
static, time dependent, non-linear or changing behaviors.

[0257]The functional phenotype of vascular endothelium can be responsive
(e.g., dynamically) to an array of physiological and pathophysiological
stimuli al of which represent types of dynamic conditions as per FIGS.
17A and 17B. Such stimuli can include biochemical substances such as
inflammatory cytokines, growth factors, circulating hormones and
bacterial products. In addition, endothelium is exposed to a number of
biomechanical stimuli resulting from the pulsatile flow of blood within
the branched vascular tree including frictional forces, fluid shear
stresses, cyclic strains (stretch) and hydrostatic pressures or the like
(yet additional types of dynamic conditions as per FIGS. 17A and 17B).

[0258]Shear stress stimulates a myriad of intracelluar events (e.g.,
intracellular signaling events) in endothelial cells which also represent
types of dynamic conditions of FIGS. 17A and 17B. Some of these events,
such as changes in intracellular calcium, protein phosphorylation and
acute stimulation of nitric oxide production, occur within seconds after
the onset of shear and other changes such as cell shape and gene
expression, occur over hours to days.

[0259]Embodiments of the system can model vascular diseases using various
types of dynamic conditions. For example, the interplay between
hemodynamic stimuli and the functional phenotype of endothelium can
affect a variety of vascular diseases.

[0260]One exemplary set of biomechanical and intracellular signaling
dynamic conditions in endothelial cells can be for atherosclerosis.
Atherosclerosis is a progressive disease, and changes within the arterial
endothelium, such as an increased permeability to lipoproteins,
endothelial cell damage and/or repair, and the expression of leukocyte
adhesion molecules can be demonstrated in the atherosclerotic process.
Interactions between apoptosis signaling kinase 1 (ASIK1), Txnip, which
is a molecule whose levels correlate with the degree of shear stress, and
thioredoxin in endothelial cells can be related to shear stress. Txnip
binds to catalytic cysteines of thioredoxin to reduce thioredoxin
activity and its ability to bind to ASK1. See, for example, Blood Flow
and Vascular Gene Expression: fluid shear stress as a modulator of
endothelial phenotype, Topper, J. N., and Gimbrone Jr., M. A., Molecular
Medicine Today, January 1999, pp. 40-46; the contents of which are
incorporated herein by reference.

[0261]Additional events that can be modeled include interactions of
endothelial cells and the types of dynamic conditions measured and/or
controlled by systems 1 and 1101 can include nitric oxide production,
enhanced expression of antioxidant enzymes like superoxide dismutase and
glutathione peroxidase or glutathione.

[0262]In one embodiment of the invention, a specimen 12 such as tubular
structure 1112 with endothelial cells is placed in a specimen holder 10
in pressure flow loop subsystem 1105, while the dynamic condition of
shear stress is controllable varied and detection of ASK1, thoioredoxin
and Txnip are monitored by systems, specimens or fluid sensors. In the
endothelial cells, shear stress associated to thoioredoxin can affect
activation of ASK1. In the absence of shear stress, thioredoxin is bound
by Txnip and maintained in an inactive state, which leads to increased
activation of ASK1. This leads to increased expression of the vascular
cell adhesion molecule 1 (VCAM1), which promotes leukocyte adhesion,
inflammation and atherosclerosis. For example, cytokine TNF-α can
lead to phosphorylation and activation of ASK1, and the activated ASK1
activates downstream MAP kinases and ultimately p38 and Jun-terminal
kinase (JNK), which increases VCAM1.

[0263]However, in the presence of shear stress, Txnip can be reduced,
liberating thioredoxin and leading to increased binding of thioredoxin to
ASK1 and inhibition of ASK1 (e.g., less ASK1 activation by TNF-α).
Thus, shear stress can be controllably set between 0 and a maximum value
in a series of steps while data is collected according one embodiment.
Then, results can be related to corresponding levels of the intercellular
activity by, for example, controller 70 or 1103.

[0264]As described above, such interrelationships between dynamic
conditions illustrate exemplary modeling targets for disclosed
embodiments. Exemplary modeling that can be performed by system
embodiments or modeling embodiments are shown in FIG. 40.

[0265]Similarly, embodiments of systems and methods described herein with
respect to FIGS. 1-47 and be used for testing/training activities.
Embodiments can be used for exemplary dynamic conditions related to
testing and training as described herein.

[0266]FIG. 48 shows steps of a preferred method for producing dynamic
conditions at regions A, as well as second order dynamic conditions at a
specimen or tubular structure 12 or 1112. The method starts at step 4801,
at which dynamic conditions gj(t) (see FIG. 17), including second
order dynamic conditions and mean dynamic conditions, are measured in
vivo.

[0267]Step 4805 involves selecting a tubular structure used to train
system 1, 1101 that yields a set of dynamic conditions closest to the
dynamic conditions gj(t)s measured in step 4801. Step 4810 involves
selecting initial global dynamic conditions and/or conditions at one or
more regions A for system 1, 1110 based on the mean of the measured
dynamic conditions gj(t).

[0268]Step 4815 involves measuring a first set of resulting dynamic
conditions gj(t) at the known stable tubular structure. Any of the
methods and systems described herein (for example, sensors A, B, Cn, Dn .
. . and/or 1, 2, 3n, 4n . . . ), as well as any other methods and systems
known in the art, can be used to directly and/or indirectly measure the
resulting dynamic conditions.

[0269]Step 4820 involves perturbing the stable tubular structure in a
manner, for example, as discussed above in connection with FIGS. 45A-45C,
46, and 47A-47H, or as otherwise discussed herein. This may involve
replacing the stable tubular structure with a second perturbed tubular
structure. The stable tubular structure is preferably perturbed in a
manner which will change the set of resulting dynamic conditions,
including resulting second order dynamic conditions, to values that are
closer to the measured set of dynamic conditions. For example, a rigid
full ring (FIG. 47H) can be used to alter the resulting dynamic
conditions and second order dynamic conditions.

[0270]At step 4825, it is determined whether the resulting set of dynamic
conditions measured at step 4801 is sufficiently close to the measured
set of dynamic conditions or a desired set of dynamic conditions. If it
is, the method ends. If not, then the method jumps back to step 4715.

[0271]In the method embodiment above, it is assumed that a selected class
of dynamic conditions (e.g., FIG. 18) was determined prior to step 1.
Further, steps 2 and 3 above presumes that a plurality of systems having
different components have been trained using a plurality of initial
tubular structures. The initial systems could include systems such as
systems 1, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 1101. Further,
the known stable initial tubular structures could be any tubular
structures such as shown in FIGS. 5A-5D, 45A-45C, 46 and 47A-47H. In
addition, perturbing the known stable tubular structure can be considered
to include another embodiment modifying the global dynamic conditions in
the pressure flow loop subsystem (e.g., pressure flow loop subsystem
1105) to generate dynamic conditions at a tubular structure mounted
therein or one or more regions A on such a tubular structure.

[0272]As discussed above, communications between the controller 1105 and
sensors (e.g., A, B, . . . ; 1, 2 . . . ) can include communications
between sensors in the fluid (represented herein by numbers such as 1, 2,
. . . ) in the system (represented herein by letters A, B, . . . ) and/or
in the specimen or tubular structure represented either by numbers 1, 2,
. . . , or letters A, B, . . . , depending on whether they were part of
the system or part of the fluid that can individually transmit (t),
receive (r) or transmit and receive (tr). System sensor in system 1101
can be in the pressure flow loop subsystem 1105 including components
thereof and/or the specimen 10 and directly or indirectly coupled to each
other and to system 1101 including controller 70 to detect dynamic
conditions (e.g., FIGS. 17A and 17B). Sensors in the fluid or fluid
sensors are referred to herein from time to time as fluid transmitters,
fluid receivers, fluid transmitter/receivers and/or fluid detectors. For
example, such fluid sensors can include nanoparticles such as nanosensors
and/or mems sensors which are indicated as 1n, 2n . . . rather than 1, 2,
. . . . Exemplary transmitter, receiver or transmitter/receiver
nanoparticles (system, fluid and/or specimen) can include a
nanotransmitter, nanoreceiver or a nanosensor or a
nanotransmitter/receiver.

[0274]Such transmitting or receiving relationships can exist for a
transmitter or a receiver. A transmitter (system, fluid and/or specimen)
is capable of sending information to a transmitter/receiver (system,
fluid and/or specimen), a receiver (system, fluid and/or specimen), a
receiving nanoparticle, a sensor or the like, a plurality of the same,
individual items above or combinations thereof. Further, the transmitter
is capable of sending information to one or more second receivers (e.g.,
system or fluid) and/or one or more third receivers (e.g., system or
fluid). Similarly, a plurality of first transmitters (e.g., system or
fluid) can each be capable of sending information to a single or
designated receiver or sensor.

[0275]A receiver (e.g., system or fluid) is capable of receiving
information from a fluid transmitter/receiver, a transmitter/receiver, a
fluid transmitter, a transmitter, a transmitting nanoparticle, a sensor,
a fluid sensor or the like, a plurality of the same, individual items
above or combinations thereof. Further, the receiver is capable of
receiving information from one or more second transmitters (e.g., system
or fluid) and/or one or more third transmitters (e.g., system or fluid).
A plurality of first receivers (e.g., system or fluid) can each be
capable of receiving information from a single or designated transmitter
or sensor.

[0276]A transmitter/receiver nanoparticle (e.g., system or fluid) is
capable of sending and/or receiving information to/from a
transmitter/receiver, a fluid transmitter/receiver, a
transmitter/receiver nanoparticle, a transmitter, a fluid transmitter, a
receiver, a fluid receiver, a nanoreceiver, nanotransmitter, a fluid
sensor, a sensor or a nanosensor or the like, a plurality of the same,
individual items above or combinations thereof. A first transmitting
and/or receiving nanoparticle (e.g., system or fluid) is capable of
sending or receiving information to/from one or more second transmitting
and/or receiving nanoparticles and/or one or more third transmitting
and/or receiving nanoparticles or more. A plurality of first
transmitter/receiver nanoparticles is capable of sending or receiving
information to/from a single or designated transmitter/receiver
nanoparticle.

[0277]Such transmitting or receiving relationships can exist for a
nanotransmitter or a nanoreceiver. A nanotransmitter (e.g., system or
fluid) is capable of sending information to a nanotransmitter/receiver, a
fluid nanoreceiver, a receiver, a fluid receiver, a fluid sensor, a
sensor, a nanosensor, a nanoreceiver or the like, a plurality of the
same, individual items above or combinations thereof. Further, a first
nanotransmitter is capable of sending information to one or more second
receiver nanoparticles (e.g., system or fluid) and/or one or more third
receiver nanoparticles (e.g., system or fluid) or more. Similarly, a
plurality of first nanotransmitters (e.g., system or fluid) can each be
capable of sending information to a single or designated nanoreceiver or
sensor (e.g., system or fluid).

[0278]A nanoreceiver (e.g., system or fluid) is capable of receiving
information from a fluid nanotransmitter/receiver, a
nanotransmitter/receiver, a transmitter/receiver, a fluid
transmitter/receiver, a fluid transmitter, a transmitter, a
nanotransmitter, a sensor or the like, a plurality of the same,
individual items above or combinations thereof. A first receiver
nanoparticle is capable of receiving information to/from one or more
second transmitter nanoparticles (e.g., system or fluid) and/or one or
more third transmitter nanoparticles (e.g., system or fluid) or more. A
plurality of first nanoreceivers (e.g., system or fluid) can each be
capable of receiving information from a single or designated
nanotransmitter or a sensor (e.g., system or fluid).

[0279]Exemplary communications between sensors can be supported by
described embodiments. For example, a transmitter/receiver (or a separate
transmit sensor and receiver sensor) is capable of transmitting
information to or transmitting/receiving information to/from a first
transmitter/receiver (or a plurality of first transmitter/receivers) and
a second transmitter/receiver (or a plurality of second
transmitter/receivers) is capable of receiving information from the first
transmitter/receiver (or the plurality of first transmitter/receivers)
and transmitting information to the transmitter/receiver. In this case,
the transmitter/receivers (e.g., system or fluid) can be transmitters or
receivers and can be nanoparticles (e.g., system or fluid) or
combinations thereof.

[0280]In another example supported by disclosed embodiments, a
transmitter/receiver is capable of receiving information from or
transmitting/receiving information to/from a first transmitter/receiver
(or a plurality of first transmitter/receivers) and a second fluid
transmitter/receiver (or a plurality of second transmitter/receivers) is
capable of transmitting information to the first fluid
transmitter/receiver (or the plurality of first transmitter/receivers)
and receiving information from the transmitter/receiver. In this case,
the transmitter/receivers (e.g., system or fluid) can be transmitters or
receivers and can be nanoparticles (e.g., system or fluid) or
combinations thereof.

[0281]A transmitter or receiver is capable of transmitting information to
or transmitting/receiving information to/from a plurality of first
transmitters or receivers and a plurality of second transmitters or
receivers is capable of respective communications (e.g., 1-to-1,
many-to-1, 1-to-many, many-to-many, 1-to-all, all to-1, all-to-all, or
the like) with each of the first transmitters or receivers to then
transmit information to the transmitter or receiver. A
transmitter/receiver is capable of receiving information from or
transmitting/receiving information to/from at least one first fluid
transmitter/receiver, and a plurality of second fluid
transmitter/receivers are capable of receiving information from the first
fluid transmitter/receiver and transmitting information to at least one
third fluid transmitter/receiver that can transmit information to the
transmitter/receiver or vice versa. Further, a system 1, 1101 could
include four or more plurality of sensors in respective communications
(e.g., 1-to-1, many-to-1,1-to-many, many-to-many, 1-to-all, all to-1,
all-to-all, or the like) with subsets or corresponding ones of each other
pluralities to transmit respective information fi(t), fj(t) or
FBj(t) therein. Again, sensors (e.g., system or fluid) can be
nanoparticles (e.g., system or fluid) or combinations thereof.

[0282]Applications of Systems

[0283]Applications for embodiments of the system broadly described herein
are numerous and varied. Although the exemplary discussion set forth
herein has focused mainly on biological applications for embodiments of
the system, and more particularly, on hemodynamic forces which act on
blood flowing through blood carrying vessels, it is well understood that
embodiments of the system may be used to reproduce any dynamic pressure
and flow environment which would benefit from the ability to
independently control pressure and flow, including both biological and
non-biological applications. For example, embodiments of the system may
be used to produce a biological environment which would simulate a number
of other in vivo conditions. These conditions may include, for example,
pressure and flow conditions within joints or on various
bone/tendon/musculature structure. Embodiments of the system may also be
used to reproduce conditions within, for example, the stomach,
intestines, esophagus, lungs, or sinus cavities, or any other such
dynamic in-vivo-environment in which such a reproduction of actual
conditions would prove beneficial. Embodiments of the system may, with
the proper cellular structure, seeding, and corresponding media, be used
to initiate and grow replacement bone/cartilage/organ structure and the
like.

[0284]Non-Biological Applications

[0285]In addition to these dynamic in-vivo conditions, certain systems as
embodied and broadly described herein may also be used to reproduce
dynamic pressure and flow environments to which non-biological elements
are subjected during operations. Such non-biological applications may
include, for example, dynamic pressure and flow through rigid/flexible
pipes/tubes in a whole host of different systems. These systems may
include, but are not limited to, for example, petroleum pipelines, fuel
flow lines in a variety of different systems, hydraulic systems,
lubrication systems, fluid lines in manufacturing facilities, and
especially those under pressure, drainage systems and related storm water
and sewage treatment systems, and any other such practical/industrial
application which would stand to benefit from such a modeling of actual
conditions.

[0286]Biological Applications

[0287]Applications in the pharmaceutical, biotechnology, life science,
academic, and research industries for a system as embodied and broadly
described herein include, but are not limited to, therapeutic screening &
testing and discovery & development of drugs, active biomolecules,
regenerative medicines, medical devices, cell & tissue devices or
therapies, drug delivery systems, personalized medicine,
genomics/proteomics, small chemical, biologics, and the like. Embodiments
of the system can be adapted to serve as a model for cardiovascular and
related pathologies such as, for example, cancer or diabetes, via
simulation of pathologic (or non-pathologic) hemodynamics that induce a
consequent pathologic (or non-pathologic) response and phenotype on
vascular cells as well as other cells. Therapies can then be designed,
developed, and tested against the pathologic model. This dynamic cell and
tissue culture environment captures in vivo phenotype, function, and
physiology more closely than traditional static cultures.

[0288]Embodiments of the system can enhance and reduce therapeutic
discovery times by providing a cost-effective platform to perform typical
and novel cell, molecular biological, and pharmacological research and
development. Embodiments of the system not only provides a means of
studying hemodynamics in normal and diseased states, but can also be used
for tissue engineering and regeneration, such as, for example, to test or
train the function of bypass vessels prior to coronary bypass surgery or
peripheral arteries or AV-shunts, or to investigate cryopreserved vessels
or for research or medical use. Example applications include
atherosclerosis, plaques (vulnerable plaque, protruding, calcified, soft,
etc.), inflammation (i.e. leukocyte adhesion), restenosis, cancer
(metastasis-tumor spreading to other tissues, extravasation-tumor and
leukocyte adhesion, and the like), and diabetes (retinopathy, blindness,
and the like).

[0289]Embodiments of the system can also provide essential capabilities
and high-throughput abilities to the pharma/biotech industries,
cell-based screening and testing, drug discovery and development, and the
like. Cell-based assays inherently evaluate test compound activity in a
biologically relevant context, with the added potential for extraction of
high information content. Embodiments of the system can be used to
systematically screen and test vast numbers of compound combinations,
testing their effects using cell-lines or primary-cell or stem cell or
patient specific stem cell cultures to allow interaction with complex
biological pathways that cannot be replicated in a cell-free assay. Other
multiple cell or tissue types can be added to certain systems such as
hepatocytes, renal, cardiac, etc. for various purposes such as providing
a test or growth environment that is representative of in-vivo
environments.

[0290]Embodiments of the system may be used in identifying potential
chemical inhibitors or activators of enzymes, receptors, or any proteins
which have effects upon cell phenotype. One method generally employs two
cell lines, preferably alike except for their expression (production) of
the protein of interest at different levels (and any further differences
necessitated by that difference in expression). Inhibitors or activators
are identified by their greater effect on the phenotype of the higher
producing cell line.

[0291]Any phenotypic characteristic of the cell which is affected by
expression of the protein of interest, other, of course, than the level
of the protein itself, may be assayed. The phenotypic characteristic is
preferably a "cultural" or "morphological" characteristic of the cell.
For purposes of this application, these terms may be defined as follows.

[0292]Cultural characteristics include, but are not limited to, the
following: the nutrients required for growth; the nutrients which, though
not required for growth, markedly promote growth; one or more physical
conditions (temperature, pH, gaseous environment, osmotic state, and
anchorage dependence or independence) of the culture which affect growth;
and the substances which inhibit growth or even kill the cells.

[0293]Morphological characteristics include, but are not limited to, the
following: the size and shape of cells; their arrangements; cell
differentiation; and subcellular structures.

[0294]Where the protein of interest is implicated in tumorigenesis or
related phenomena, the characteristic observed is preferably one related
to cellular growth control, differentiation, de-differentiation,
carcinogenic transformation, metastasis, tumorigenesis, or angiogenesis.

[0295]Phenotypic changes which are observable with the naked eye are of
special interest. Changes in the ability of the cells to grow in an
anchorage-independent manner, to grow in soft agar, to form foci in cell
culture, and to take up selected stains, for example, are particularly
appropriate phenomena for observation and comparison.

[0296]The higher producing cell line is preferably obtained by introducing
a gene encoding the Protein of Interest (POI) into a host cell or, if a
native protein of the cell, by introducing a promoter into the cellular
genome upstream of and operatively linked to the POI. The gene may be a
one isolated from the genome of an organism, a cDNA prepared from an mRNA
transcript isolated from an organism, or a synthetic duplicate of a
naturally occurring gene. It may also have a sequence which does not
occur exactly in nature, but rather corresponds to a mutation (single or
multiple) of a naturally occurring sequence (also referred to as a
"wild-type sequence"). No limitation is intended on the manner in which
this mutated sequence is obtained.

[0297]The gene is preferably operably linked to a promoter of gene
expression which is functional in the host, such that the corresponding
POI is stably "overproduced" in the recipient cells to differing degrees.
The promoter may be constitutive or inducible. By "overproduced", it is
meant that the POI is expressed at higher levels in the genetically
manipulated cell line than in the original cell line. This allows one to
generate cell lines which contain (or secrete) from as little as a few
fold to more than 100-fold elevated levels of the POI relative to the
control cells.

[0298]Any method may be used to introduce the gene into the host cell,
including transfection with a retroviral vector, direct transfection
(e.g., mediated by calcium phosphate or DEAE-dextran), and
electroporation. Preferably, a retroviral vector is used

[0299]The host cells should exhibit a readily observable phenotypic change
as a result of enhanced production of the POI. Preferably, this response
should be proportional to the level of production of the POI. Finally,
the cells should not spontaneously manifest the desired phenotypic
change. For example, 3T3 cells form foci spontaneously. Among the
preferred cell lines for these methods are Rat-6 fibroblasts, C3H10T 1/2
fibroblasts, and HL60. (HL60 is a human cell line that differentiates in
response to PKC activation.) 3T3 cells may be used, but with the
reservation stated above.

[0300]Generally speaking, it is preferable to maximize the ratio of
production by the "overproducing" cell line to production by the "native"
line. This is facilitated by selecting a host cell line which produces
little or no POI, and introducing multiple gene copies and/or using high
signal strength promoters.

[0301]The Rat 6 embryo fibroblast cell line is a variant of the rat embryo
fibroblast cell line established by Freeman et. al., (1972) and isolated
by Hsiao et al., 1986. This cell line has an unusually flat morphology,
even when maintained in culture at post-confluence for extended periods
of time, displays anchorage dependent growth and, thus far, has not
undergone spontaneous transformation. It was also ideal for these studies
since it has a very low level of endogenous PKC activity and a low level
of high affinity receptors for phorbol esters.

[0302]According to these methods, one looks for is a increase in the
phenotypic change exhibited by the cell which becomes greater with
increasing expression of the POI. This is a "graded cellular response,"
and it is by this specialized response that inhibitors or activators of
the POI can be distinguished from agents that act upon other cell
metabolites to effect a phenotypic change.

[0303]Thus, in a preferred embodiment, the cell lines are assayed for
their relative levels of the POI, and their ability to grow in
anchorage-independent systems (e.g., matrices such as soft agar or
methocel), to form small "foci" (areas of dense groups of cells clustered
together and growing on top of one another) in tissue culture dishes, to
take up selected stains, or to bind an appropriately labeled antibody or
other receptor for a cell surface epitope. In addition to exhibiting
these growth control abnormalities, such cell lines will also be
sensitive in their growth properties to chemical agents which are capable
of binding to, or modifying the biological effects of, the POI.

[0304]In selected embodiments, the method is particularly unique in that
it can be employed to search rapidly for EITHER activators OR inhibitors
of a given POI, depending upon the need. The term "activators," as used
herein, includes both substances necessary for the POI to become active
in the first place, and substance which merely accentuate its activity.
The term "inhibitors" includes both substance which reduce the activity
of the POI and these which nullify it altogether. When a POI has more
than one possible activity. The inhibitor or activator may modulate any
or all of its activities.

[0305]The use of this screening method to identify inhibitors or
activators of enzymes is of special interest. In certain preferred
embodiments, the method is used to identify inhibitors or activators of
enzymes involved in tumorigenesis and related phenomena, for example,
protein kinase C, omithine decarboxylase, cyclic AMP-dependent protein
kinase, the protein kinase domains of the insulin and EGF receptors, and
the enzyme products of various cellular one genes such as the c-src or
c-ras genes.

[0307]With the exception of the brain, where its expression is very high,
PKCbeta-1 is expressed at veer low levels in most tissues, and its
expression is virtually undetectable in Rat 6 fibroblasts (see below).
Thus, using this form will maximize the phenotypic differences observed
between control cells and cells overexpressing the introduced form of
PKC. The PKCbeta-form is also of particular interest because within the
PKC gene family its deduced carboxy terminal domain displays the highest
overall homology to the catalytic subunit of the cyclic AMP-dependent
protein kinase (PKAc) and the cyclic GMP-dependent protein kinase (PKG)
(Housey et al., 1987). The latter observation suggests that PKAc, PKG,
and the beta-1 form of PKC may share a common ancestral serine/threonine
protein kinase progenitor, and that the additional PKC genes may have
been derived through evolutionary divergence from the beta-1 form.

[0308]Agents that interact with certain structural proteins, such as actin
and myosin, are also of interest. Mutations in the genes expressing these
proteins may be involved in tumorigenesis and metastasization. Such
interactions can lead to changes in cell phenotype which can be assayed
by this method.

[0309]In additional studies with other genes, most notably the c-H-ras
oncogene, the catalytic subunit of the cyclic AMP-dependent protein
kinase, the c-myc oncogene, and certain cDNA clones encoding
phorbol-ester inducible proteins, similar results have been obtained.
Thus it is also clear that the method can be generalized to a vide
variety of genes encoding proteins which are involved in cellular growth
control in numerous cell types.

[0310]One embodiment of a preferred protein inhibitor/activator drug
screening method of the invention can include the following steps:

[0311]1. Construction of an expression vector which is capable of
expressing the protein of interest in the selected host by inserting a
gene encoding that protein into a transfer vector. The gene may be
inserted 3' of a promoter already borne by the transfer vector, or a gene
and a promoter may be inserted sequentially or simultaneously.

[0312]2. Introduction of the expression vector (a) into cells which
produce recombinant retrovirus particles, or (b) directly into host cells
which will be used for subsequent drug screening tests (the resulting
cells are called herein "test" cells). In parallel, the transfer vector
(i.e., the vector lacking the gene of interest and possibly a linked
promoter but otherwise identical to the expression vector) is preferably
also introduced into the host cells. Cell lines derived from this latter
case will be used as negative controls in the subsequent drug screening
tests. Alternatively, the unmodified host cells may be used as controls.

[0313]If (a) was employed above, after an appropriate time (e.g., 48
hours), media containing recombinant virus particles is transferred onto
host cells so as to obtain test or control cells.

[0314]3. The test and control cells are transferred to selective growth
medium containing the appropriate drug which will only allow those cells
harboring the expression vector containing the selectable marker gene (as
well as the gene or cDNA of experimental interest) to grow. After an
appropriate selection time (usually 7-10 days), individual clones of
cells (derivative cell lines) are isolated and propagated separately.

[0315]4. Each independent cell line is tested for the level of production
of the POI. By this method, a range of cell lines is generated which
overproduce from a few fold to more that 100-fold levels of the POI. In
parallel, the control cell lines which contain only the transfer vector
alone (with the selectable marker gene) are also assayed for their
endogenous levels of the POI.

[0316]5. Each independent line is then tested for its growth capability in
soft agar (or methocel, or any other similar matrix) of various
percentages and containing different types of growth media until cell
lines are identified which possess the desired growth characteristics as
compared to the control cell lines.

[0317]6. Each cell line is also tested for its ability to form "foci", or
areas of dense cellular growth, in tissue culture plates using media
containing various percentages and types of serum (20%, 10%, 5% serum,
fetal calf serum, calf serum, horse serum, etc.) and under various
conditions of growth (e.g. addition of other hormones, growth factors, or
other growth supplements to the medium, temperature and humidity
variations, etc.). In these tests, the cells are maintained at
post-confluence for extended periods of time (from two to eight weeks)
with media changes every three days or as required. Such growth
parameters are varied until cell lines are identified which possess the
desired foci formation capacity relative to the control cell lines under
the identical conditions.

[0318]7. After a cell line possessing the required growth characteristics
is identified, the cells are grown under the conditions determined in (5)
above with the growth medium supplemented with either crude or purified
substances which may contain biologically active agents specific to the
POI. Thus, crude or purified substances possessing the latter properties
can be rapidly identified by their ability to differentially alter the
growth properties of the experimental cells (which overproduce the POI)
relative to the control cells (which do not). This can be done rapidly
even by simple observation with the naked eye, since the colonies which
grow in soft agar after 2 weeks are easily seen even without staining,
although they may be stained for easier detection.

[0319]Similarly, if the post-confluence foci formation assay is chosen,
the foci which result after approximately two weeks can be easily seen
with the naked eye, or these foci can also be stained. Results of the
assays can be rapidly determined by measuring the relative absorbance of
the test cells as compared to the control cells (at 500 nm, or another
appropriate wavelength). In this fashion, thousands of compounds could be
screened per month for their biological activity with very low labor and
materials costs.

[0320]Furthermore, if antigen expression varies on the test cells
expressing high levels of the POI relative to the control cells, a simple
Enzyme-Linked Immunoadsorption Assay (ELISA) could be performed and an
antibody specific to the antigen.

[0321]While the assay may be performed with one control cell line and one
test cell line, it is possible to use additional lines, tests lines with
differing POI levels. Also additional sets of control/test lines,
originating from other hosts, may be tested.

[0322]The system can also be used for identifying agents that bind to
cellular targets, such as membrane proteins, but without necessarily
affecting or altering the phenotype of the cell.

[0323]For example, the system may be used determine the ability of an
agent to bind to a particular site on a membrane protein and thereby
alter the level of surface expression thereof. Such an alteration in
surface expression may result from the agent blocking a site on the
mutant that corresponds to an active site on the wild-type membrane
protein and/or by blocking intracellular trafficking and/or processing of
the mutant membrane protein. Alternatively, an alteration in surface
expression may result from the agent improving intracellular trafficking
of the mutant membrane protein.

[0324]As described above, there are a wide variety of formats known and
available to those skilled in the art for appropriate binding assays.
According to certain embodiments of the invention, one or more cells
expressing a membrane POI may be provided in a suitable liquid medium and
exposed to one or more candidate compounds, while in other embodiments
the cells may be immobilized on a surface and then exposed to the
candidate compound(s). Similarly, according to still other embodiments of
the invention, one or more candidate compounds may be immobilized on a
surface and exposed to a liquid medium containing one or more cells that
express a membrane protein of interest or the candidate compound(s) may
be included in a suitable liquid medium to which one or more cells
expressing a membrane protein of interest is added.

[0325]Binding is often easier to detect in systems in which at least one
of the candidate compound and the membrane POI is labeled (e.g., with
fluorescence, radioactivity, an enzyme, an antibody, etch, including
combinations thereof, as known to those skilled in the art). After
exposing the candidate compound to the cell expressing a membrane protein
and washing off or otherwise removing unbound reagents, the presence of
the labeled moiety (i.e., bound to the unlabelled component of the test
system) is measured.

[0326]Methods for performing various binding assays are known in the art,
including but not limited to the assay systems such as those described in
PCT Application US98/18368. Various references provide general
descriptions of various formats for protein binding assays, including
competitive binding assays and direct binding assays, (see e.g., Stites
and Terr, Basic and Clinical Immunology, 7th ed. (1991); Maggio, Enzyme
Immunoassay, CRC Press, Boca Raton, Fla. (1980); and Tijssen, Practice
and Theory of Enzyme Immunoassays, in Laboratory Techniques in
Biochemistry and Molecular Biology, Elsevier Science Publishers, B.V.
Amsterdam, (1985)).

[0327]Thus, according to certain embodiments, immunoassays are provided in
which one or more cells expressing a membrane protein of interest are
generally bound to a suitable solid support and combined with a candidate
agent, and observing changes in the level of surface expression of the
membrane POI. In these embodiments, one or more of the assay components
is attached to a solid surface.

[0328]In some embodiments, an assay system may used (as known in the art)
to detect the change in the surface expression of the membrane protein
due to the binding of the candidate agent. For example, if the membrane
protein of interest is a membrane ion channel, a patch clamp assay may be
employed to detect a change in the flux of ions across the membrane, thus
evidencing an increase in the level of surface expression of the ion
channel.

[0329]In alternative embodiments, an indirect immunoassay system is used
in which the membrane protein on the surface of the cell(s) is detected
by the addition of one or more antibodies directed against an
extracellular epitope of the membrane protein, as known in the art.

[0330]When using a solid support in embodiments of methods according to
the invention, virtually any solid surface is suitable, as long as the
surface material is compatible with the assay reagents and it is possible
to attach the component to the surface without unduly altering the
reactivity of the assay components. Those of skill in the art recognize
that some components exhibit reduced activity in solid phase assays, but
this is generally acceptable, as long as the activity is sufficient to be
detected and/or quantified. Suitable solid supports include, but are not
limited, to any solid surface such as glass beads, planar glasses,
controlled pore glasses, plastic porous plastic metals, or resins to
which a material or cell may be adhered, etc.). Those of sill in the art
recognize that in some embodiments, the solid supports used in the
methods of the invention may be derivatized with functional groups (e.g.,
hydroxyls, amines, carboxyls, esters, and sulfhydryls) to provide
reactive sites for the attachment of linkers or the direct attachment of
the candidate agent or other assay component.

[0332]In some embodiments, standard direct or indirect ELISA, IFA, or RIA
methods as generally known in the art are used to detect the binding of a
candidate agent to a membrane POI. In some embodiments, an increase in
the level of surface expression of the membrane protein is detected in a
sample; while in other embodiments, a decrease in the level of surface
expression is detected. Thus, it is clear that embodiments of methods
according to the invention are adaptable to the detection,
identification, and characterization of multiple elements.

[0333]Accordingly, in some particularly preferred embodiments of the
methods of the invention, a sandwich ELISA (enzyme-linked immunosorbent
assay) with a monoclonal or polyclonal antibody for capture ("a capture
antibody") and a secondary antibody ("a reporter antibody") for detection
of bound antibody-antigen complex may be used.

[0334]In some preferred ELISA embodiments, alkaline phosphatase conjugates
are used, while in still other preferred embodiments, horseradish
peroxidase conjugates are used. In addition, avidin/biotin systems may
also be used, particularly for assay systems in which increased signal
are desired. Suitable enzymes for use in preferred embodiments include,
but are not limited to, peroxidases, luciferases, alkaline phosphatases,
glucose oxidases, beta-galactosidases and mixtures of two or more
thereof.

[0335]In addition to the assay systems in which a solid support is
utilized, the invention pro-ides embodiments of methods in which the
assay components remain suspended in solution.

[0336]Any change, such as an increase or decrease, in the level of binding
in the presence of the candidate agent relative to control indicates that
the candidate agent alters the level of surface expression of the first
mutant form of the membrane protein.

[0337]The determination of the level of surface expression of the integral
membrane protein of interest may be performed using any of the methods
and techniques known and available to those skilled in the art.
Preferably, the level of binding is determined by fluorescence,
luminescence, radioactivity, absorbance or a combination of two or more
of these.

[0338]According to certain embodiments of the invention, the extracellular
epitope to which the antibody binds on the membrane protein is preferably
the same as a wild-type epitope, i.e. an extracellular epitope found on
the naturally-occurring form(s) of the membrane protein of interest.
Without wishing to be bound to any theory of operability or the line,
such an arrangement may have the potential to reduce errors arising from
differences in protein structure, for example by a change in one or more
of the functional properties of the protein.

[0339]According to certain embodiments of the invention, the extracellular
epitope may also contain a tag. Suitable tags are known and available to
those skilled in the art. A particularly preferred tag for use in
selected methods of the invention is a hemagglutinin (HA) tag. The tag
may be inserted in an extracellular domain of the POI or may replace a
portion of an extracellular domain thereof.

[0340]A method of identifying an agent that alters the level of surface
expression or binds to an extracellular epitope of a membrane protein in
a mammalian cell according to disclosed embodiments can include preparing
a first medium containing mammalian cells that express the membrane
protein, adding to the first medium containing mammalian cells an
effective amount of a candidate agent, incubating the cells in the
presence of the candidate agent for a sufficient period of time in a
system according to embodiments, adding to the first medium containing
mammalian cells an effective amount of at least one antibody which binds
to at least one extracellular epitope of the membrane protein and
determining the level of binding of the at least one antibody to the
extracellular epitope following incubation with the candidate agent,
wherein a change in the level of binding relative to control indicates
that the candidate agent alters the level of surface expression of the
membrane protein or binds to the extracellular epitope of the membrane
protein.

[0341]A method for detecting the presence of a protein or gene of interest
in a sample, according to one embodiment can include (a) placing a sample
in a system according embodiments, (b) contacting the sample with a
compound which selectively hybridizes to the gene of interest or binds to
the protein and (c) determining whether the compound hybridizes to the
gene of interest or binds to the protein in the sample.

[0342]A method for identifying a compound which binds to or modulates the
activity of a protein according to one embodiment can include (a)
immobilizing a cell expressing the protein in a system according
embodiments, (b) contacting the cell with a test compound and (c)
determining whether the protein binds to the test compound or determining
the effect of the test compound on the activity of the protein.

[0343]A method of identifying a nucleic acid molecule associated with a
disorder or identifying a subject having a disorder or at risk for
developing a disorder according to embodiments of the invention can
include (a) placing a sample containing nucleic acid molecules from a
subject with or at risk of developing a disorder in a system according
embodiments, (b) contacting the sample with a hybridization probe that
contains a nucleic acid sequence indicative of the disorder or risk for
developing the disorder and (c) detecting the presence of a nucleic acid
molecule in the sample that hybridizes to the probe, thereby identifying
a nucleic acid molecule associated with a disorder or the subject having
the disorder or at risk for developing the disorder.

[0344]In accordance with another embodiment, a pharmaceutical composition
can include a therapeutically effective amount of an agent identified or
described herein and a pharmaceutically effective carrier.

[0345]In accordance with another embodiment, a method can include adding
an effective amount of at least one primary antibody and an effective
amount of at least one secondary antibody, wherein the primary antibody
binds to an extracellular epitope of a membrane protein and the secondary
antibody binds to the first antibody. In accordance with another
embodiment, a level of binding can be measured by fluorescence,
luminescence, radioactivity, absorbance or a combination of two or more
thereof.

[0346]In accordance with another embodiment, an integral membrane protein
can be a membrane ion channel. In accordance with another embodiment, a
membrane ion channel can be a sodium channel, a potassium channel, a
calcium channel or a chloride channel.

[0347]In accordance with another embodiment, a primary or secondary
antibody can be coupled to an enzyme. In accordance with another
embodiment, an enzyme can be selected from the group including or
consisting of peroxidases, luciferases, alkaline phosphatases, glucose
oxidases, beta-galactosidases, or the like and mixtures of two or more
thereof.

[0348]Other applications in the tissue regeneration and engineering,
clinical, and research industries include, but are not limited to, tissue
engineering arteries and veins for cardiovascular repair or replacement
and training ex vivo veins or arteries prior to implantation or applying
a treatment to the specimen prior to implantation. For example,
embodiments of the system can simulate complex coronary hemodynamics for
growing tissue engineered or regenerated arteries and for training
saphenous veins or defrosting cryogenic arteries in preparation for the
harsh and dynamic coronary environment or other peripheral arterial
disease regions. Another example may include treating an artery or vein
with gene, RNAi, or other biomolecular therapy in conjunction with
hemodynamic simulation prior to therapeutic intervention. Embodiments of
the system can provide accurate and precise control of physiologic
parameters for applications in the tissue regeneration and engineering
industries. For example, to grow vascular grafts seeded with stem cells,
hemodynamic stimuli ranging from coronary to peripheral arteries, as well
as biochemical stimuli, such as growth factors, can be applied to
condition the stem cells to differentiate to vascular cells that are
preferable functional.

[0349]The surfaces of synthetic vascular prostheses are capable of
provoking platelet activation and blood coagulation, generating clots
that can rapidly occlude the engrafted prosthetic. Thus, the field of
synthetic vascular grafts has developed at a cautious pace, and efforts
to ensure their safety have included the testing of different graft
materials and the inclusion of anti-thrombogenic materials in the
pre-treatment used to seal the interstices of the graft to prevent blood
loss from the vessel. (Sausage, L. R., in Haimovici et al., eds.,
Haimovici's Vascular Surgery, 4th ed., 1996). Today, only polyethylene
terephthalate (DACRON) and polytetrafluoroethylene (TEFLON) are approved
by the Federal Drug Administration for this use.

[0350]Even so, autologous grafts still are considered superior to
synthetic ones because their endothelial linings, which secrete a number
of natural anti-thrombotic substances, provide a far better flow surface
than the material used for today's synthetic prostheses. Unfortunately,
only a limited number of the body's blood vessels provide tissue suitable
for use in autologous vascular transplants, and improvements in the field
of synthetic prostheses would prove a boon to many patients, especially
those requiring multiple heart bypasses.

[0351]Another limitation of synthetic vascular prostheses currently
approved for use is that the caliber, i.e., inner diameter, of grafts
deemed as acceptable must be at least 6 mm. It is believed, in fact, that
no satisfactory synthetic prosthesis having a caliber below 6 mm exists
today (e.g., Sauvage, 1996). Thus, the need for smaller caliber grafts
remains unfulfilled, even though numerous patients require repeat
coronary bypass, or have peripheral arterial occlusions below the knee or
in the cerebrovascular tree, which would use small caliber synthetic
grafts if these were available.

[0355]One application of exemplary embodiments of systems and methods
described herein is combination (or hybrid) medical devices and cell
therapy. A hybrid or combination vascular graft is one exemplary medical
device. A hybrid vascular graft is made up of both synthetic material and
living cells. Embodiments of a hybrid or combination vascular graft will
now be described. Embodiments of a hybrid vascular graft can be developed
using exemplary embodiments of systems and methods described herein (e.g.
FIGS. 1-48), however, an embodiment of the hybrid vascular graft is not
intended to be so limited thereby.

[0356]Fin embodiment of a hybrid vascular graft can be a synthetic
vascular graft (e.g., silicon) combined with living cells (e.g., a
biomaterial) that can reduce or eliminate the need for the costly
dependence on drugs, reduce subsequent surgeries and more accurately
reflect human biology. For example, the hybrid graft embodiment can
recapitulate native function and/or the living cells can be functional
endothelial cells (e.g., evidenced by cell characteristics, expression
profiles or metabolism). The hybrid graft embodiment can replicate the
original physiologic function of living arteries and veins with vascular
cells. Further, the hybrid graft embodiment can be used for the difficult
or previously impossible small diameter synthetic grafts (e.g., 6 mm or
less, 4 mm or less). The hybrid graft embodiment can use endothelial
cells or other cells (e.g., stem cells) that differentiate into in
endothelial cells that are attached to a synthetic graft. Once cells
(e.g., endothelial cells) are attached (e.g., as conventionally known) to
the synthetic graft a functional coating (e.g., a confluent monolayer) of
cells is grown on the hybrid grafts using disclosed systems and methods
(e.g., FIGS. 36-39).

[0358]Arterial diseases include Peripheral Arterial Disease (PAD), which
is the build up of fat on the artery wall and narrowing of the artery
structure limiting blood supply and atherosclerosis. PAD can occur in
locations, such as carotid artery, renal artery, iliac artery, femoral
artery, popliteal artery, or tibial artery. Atherosclerosis is a chronic
disease in which thickening, hardening, and loss of elasticity of the
arterial walls result in impaired blood flow. In addition, vascular
failures can cause diseases including angina, high blood pressure, high
cholesterol, heart attack, stroke, and arrhythmia.

[0359]Treatment of such diseases can include bypass or graft surgery. An
exemplary double bypass graft can use one bypass to connect the internal
mammary artery to a branch of the left coronary artery and the other
bypass to connect the aorta to the right coronary artery. A major mode of
treatment for cardiovascular diseases using bypass or graft surgery is
via synthetic vascular grafts.

[0360]However, disadvantages of prosthetics or synthetic vascular grafts
include mechanical disadvantages, such as poor compliance (e.g., rigid),
size mismatch and viscoelasticity, and biocompatibility disadvantages.
Biocompatibility complications include intimal hyperplasia at
anastamoses, thrombosis, restenosis (rapid uptake), infection, bacteria
colonization, dilatation or rupture. Vascular grafts can also fail
because of compliance mismatch, such as within the bulk material, within
the sutured attachment to the existing vessel or at the anastamosis.

[0361]A hemodialysis access graft though an arterio-venous (AV) shunt is a
looped graft between an artery and a vein (e.g., in the body). For
example, the AV shunt can be located in the upper arm, middle arm, lower
arm or combinations thereof. The blood can be transferred to a dialysis
machine from the portion of the AV shunt connected to the artery and
returned from the dialysis machine to the portion of the AV shunt
connected to the vain.

[0362]Stents elicit negative reactions from the body since the material is
non-living or non-biological, and thus subsequently fail because of
re-closure of a treated blood vessel caused by growth of smooth muscle
cells, stent thrombosis, and structural/mechanical failure of the graft
or the like.

[0363]In contrast, embodiments of hybrid grafts can consider the
biophysical environment the graft will be in, such as, for example, the
cardiovascular system, including simulation of in vivo hemodynamics
(e.g., concurrent wall shear stress, stretch, and pressure). Embodiments
of the hybrid vascular graft can be processed in vitro to grow or train
endothelial cells on the vascular graft surface (e.g., tubular structure)
that can function as if it was in a desired in vivo environment. Related
art technologies cannot grow cells on a vascular graft let alone
functional endothelial cells because stretch devices produce only a
biaxial or heterogeneous strain field without applied flow, and flow
devices produce only a flow field without stretch.

[0364]Embodiments of hybrid vascular grafts can utilize/train with regard
to a physical nature of a graft or disease, a dynamic environment of a
graft and/or the dynamic nature of disease. Further, embodiments of
hybrid vascular grafts can be developed at a size greater than 6 mm, but
also at a size 6 mm or less, 5 mm or less, 4 mm or less or the like and
have significantly reduced risks or clogging or thrombosis. Such risk
reduction is achieved by training the endothelial cells or stem cells
that ultimately differentiate into endothelial cells that yields
functional endothelial cells that line the hybrid graft.

[0365]One embodiment of a hybrid graft can be developed using a synthetic
vascular graft provided with stem cells (as is known in the art, e.g.,
vascular cell origin from hemangioblast) and exposed to controlled
hemodynamics resulting in an exemplary graft with functional vascular
endothelial cells. Such an exemplary embodiment of a hybrid vascular
graft with functional vascular endothelial cells can be used as described
above. According to another embodiment, stem cells can be extracted from
the patient intended to receive the embodiment of a hybrid vascular graft
(a combination synthetic graft).

[0366]In one embodiment for preparing a hybrid vascular graft, a plurality
of cells is affixed to a surface of a synthetic graft. A binding material
(e.g., adhesion proteins, fibronectin) can be used to coat a surface of
the synthetic graft to affix the plurality of cells. In another
embodiment for preparing a hybrid vascular graft, etching of the
synthetic graft can improve surface adhesion of proteins and cells that
can reduce or remove the necessity of a binding material. Etching with
plasma treatments can include oxygen plasma, glow-discharge plasmas or
amide and amine containing plasmas. Further, for polytetrafluorethylene
(PTFE) or ePTFE, ammonia and oxygen plasmas can be used and fluorine can
be replaced with amines and nitrogen groups to help facilitate adhesion
of proteins and cells (e.g., EC).

[0367]Additional exemplary embodiments of methods for processing
biomaterials, non-biomaterials or combinations thereof (e.g., hybrid
vascular grafts) will now be described.

[0368]An exemplary method embodiment of preparing a biomaterial intended
for implantation into a mammal in need thereof can include placing the
biomaterial in a system according embodiments of the invention for a
sufficient time prior to implantation of the biomaterial into the mammal.
An exemplary method embodiment of promoting engraftment of a biomaterial
following implantation into a mammal's body can include placing the
biomaterial in a system according to disclosed embodiments prior to
implantation of the biomaterial into the mammal's body.

[0369]As shown in FIG. 49, an exemplary method of using systems disclosed
herein (e.g., systems 1, 1101) for treating or processing a biomaterial
will now be described, As shown in FIG. 49, selected biomaterials such as
cells (e.g., endothelial cells, stem cells) can be combined with a
non-biomaterial (e.g., a synthetic graft) (block 4905). The combination
can then be placed in a simulator of a selected class of dynamic
conditions (e.g., selected system embodiment 1, 1101) (block 4910). The
combination is then exposed to a selected or prescribed dynamic condition
or series of conditions (e.g., hemodynamic conditions of an abdominal
aorta) (block 4915). The combination (e.g., a hybrid synthetic graft) can
then be continuously monitored or periodically monitored for desired
results (e.g., generation of a confluent functional monolayer of
endothelial cells) or for a selected period of time (block 4920).
Optionally, the controlled dynamic conditions can be repeated or modified
(e.g., "dial-up") according to feedback (e.g., FBj(t)) from the
monitored combination or its environment or desired results (block 4925).
When the time periods have elapsed or results have been obtained, the
combination can be extracted from the selected dynamic condition class
simulator (block 4830). The modified combination can then be implanted in
a mammal. The method embodiment of FIG. 49 can be performed on
biomaterials alone.

[0370]An exemplary method embodiment of promoting endothelialization of a
vascular graft can include (a) immobilizing a plurality, of endothelial
cells on at least one surface of a vascular graft and (b) placing the
vascular graft in a system according to disclosed embodiments under
conditions effective to promote the endothelial cells to form a confluent
monolayer on the surface of the vascular graft.

[0371]An exemplary method embodiment of coating a vascular graft with a
confluent monolayer of endothelia cells can include (a) immobilizing a
plurality of endothelial cells on at least one surface of a vascular
graft; and (b) placing the vascular graft in a system according disclosed
embodiments under conditions effective to promote the endothelial cells
to form a confluent monolayer on the surface of the vascular graft. An
exemplary method embodiment of coating a vascular graft with a confluent
monolayer of endothelial cells can include (a) immobilizing a plurality
of multipotent stem cells on at least one surface of a vascular graft,
(b) placing the vascular graft in a system according to disclosed
embodiments under conditions effective to promote the stem cells to form
confluent monolayer on the surface of the vascular graft and (c) placing
the vascular graft in an environment that promotes the stem cells to
differentiate into endothelial cells.

[0372]An exemplary method embodiment of promoting endothelialization of a
vascular graft can include (a) immobilizing a plurality of multipotent
stem cells on at least one surface of a vascular graft; and (b) placing
the vascular graft in a system according to disclosed embodiments under
conditions effective to promote the stem cells to form a confluent
monolayer on the surface of the vascular graft or to differentiate into
endothelial cells on the surface of the vascular graft.

[0373]An exemplary method embodiment for the generation of tissue can
include (a) immobilizing a plurality of cells in at least one surface of
a matrix, the matrix including a suitable biomedical material; and (b)
placing the matrix in a system according to disclosed embodiments under
conditions effective to promote the cells to grow on the surface of the
matrix. An exemplary method embodiment of storing an organ prior to
transplantation into a patient in need thereof; can include placing the
organ in a system according to disclosed embodiments under conditions in
which the organ remains substantially unchanged or viable for an extended
period of time.

[0374]In accordance with another embodiment, a coating including at least
one cell is applied to at least a portion of at least one surface of the
biomaterial (e.g., vascular graft, matrix or the like) prior to placement
in the system. In accordance with another embodiment, the cell is
selected from embryonic stem cells, adult stem cells, mesenchymal stem
cells, endothelial cells, smooth muscle cells, osteocytes, or
osteoblasts. In accordance with another embodiment, the coating can
include an affixing substance selected from fibronectin, fibrin glue,
combinations of fibrinogen and thrombin, collagen, basement membrane, or
alginate, and mixtures of two or more thereof.

[0375]In accordance with another embodiment, the coating further can
include at least one supplement selected from an analgesic, an
anesthetic, an antimicrobial compound, an antibody, an anticoagulant, an
antifibrinolytic agent, an anti-inflammatory compound, an antiparasitic
agent, an antiviral compound, a cytokine, a cytotoxin or cell
proliferation inhibiting compound, a chemotherapeutic drug, a growth
factor, an osteogenic or cartilage inducing compound, a hormone, an
interferon, a lipid, an oligonucleotide, a polysaccharide, a protease
inhibitor, a proteoglycan, a polypeptide, a steroid, a vasoconstrictor, a
vasodilator, a vitamin, or a mineral, and mixtures of any two or more
thereof.

[0376]In accordance with another embodiment, a supplemented coating is
applied to the biomaterial in an amount effective to promote cell
migration, cell proliferation and/or cell differentiation in a
cell-containing environment. In accordance with another embodiment a
supplemented coating is applied to the biomaterial in an amount effective
to promote endothelialization of the biomaterial in an endothelial
cell-containing environment, where such endothelialization can cause a
confluent layer of cells to form on the surface of the biomaterial when
the biomaterial is placed into the endothelial cell-containing
environment. In accordance with another embodiment, a supplemented
coating is applied to the biomaterial in an amount effective for the
prophylaxis or treatment of infection in a patient when the biomaterial
is placed into a patient.

[0377]In accordance with another embodiment, a biomaterial can include or
combine with an orthopedic device, a urinary catheter, an intravascular
catheter, a suture, a vascular prosthesis, an intraocular lens, a contact
lens, a heart valve, a shoulder replacement device, an elbow replacement
device, a hip replacement device, a knee replacement device, an
artificial heart, a fixation plate, a dental implant, a nasal implant, a
breast implant, a testicular implant, a sponge, a film or a bag. In
accordance with another embodiment, a biomaterial can be prepared
according to such exemplary method embodiments.

[0378]In accordance with another embodiment, the biomaterial can be
combined with a synthetic vascular graft or prosthesis. In accordance
with another embodiment, the biomaterial intended for implantation into a
mammal includes a synthetic vascular graft or hybrid vascular graft.

[0379]In accordance with another embodiment, the biomaterial intended for
implantation into a mammal can be used for a hybrid hemodialysis access
graft, a hybrid femoral artery graft or a hybrid coronary bypass vascular
graft. In accordance with another embodiment, a hybrid vascular graft is
one of at least 8 mm, less than 8 mm, in less than 6 mm, less than 5 mm,
less than 4 mm less than 3 mm less than 2 mm less than 1 mm, or less than
0.5 mm in diameter.

[0380]Embodiments of the system may also be used to differentiate
undifferentiated cells, such as, for example, adult stem cells or
progenitor cells, toward a particular differentiated state such as, for
example, an adult stem cell to an endothelial cell or a smooth muscle
cell. The technology can also be used to train or condition cells or
tissue such as saphenous vein or tissue engineered artery or vein. Organs
or tissues can also be used in embodiments of the system to provide the
correct physiologic simulation for various applications such as research,
development, organ transport, or the construction of a more in vivo like
system and the like. Embodiments of the system can also be used to
maintain, grow, or enhance the growth of various organs, cells, and
tissues such as liver, kidney, heart, bone, or synovial tissue.

[0381]The most promising source of organs and tissues for transplantation
lies in the development of stem cell technology. Theoretically, stem
cells can undergo self-renewing cell division to give rise to
phenotypically and genotypically identical daughters for an indefinite
time and ultimately can differentiate into at least one final cell type.
By generating tissues or organs from a patient's own stem cells, or by
genetically altering heterologous cells so that the recipient immune
system does not recognize them as foreign, transplant tissues can be
generated to provide the advantages associated with xenotransplantation
without the associated risk of infection or tissue rejection.

[0382]Stem cells also provide promise for improving the results of gene
therapy. A patient's own stem cells could be genetically altered in
vitro, then reintroduced in vivo to produce a desired gene product. These
genetically altered stem cells would have the potential to be induced to
differentiate to form a multitude of cell types for implantation at
specific sites in the body, or for systemic application. Alternately,
heterologous stem cells could be genetically altered to express the
recipient's major histocompatibility complex (MHC) antigen, or no MHC, to
allow transplant of those cells from donor to recipient without the
associated risk of rejection.

[0383]Stem cells are defined as cells that have extensive, perhaps
indefinite, proliferation potential di-t differentiate into several cell
lineages, and that can repopulate tissues upon transplantation. The
quintessential stem cell is the embryonal stem (ES) cell, as it has
unlimited self-renewal and multipotent differentiation potential. These
cells are derived from the inner cell mass of the blastocyst, or can be
derived from the primordial germ cells from a post-implantation embryo
(embryonal germ cells or EG cells). ES and EG cells have been derived
from mouse, and more recently also from non-human primates and humans.
When introduced into mouse blastocysts or blastocysts of other animals,
ES cells can contribute to all tissues of the mouse (animal). When
transplanted in post-natal animals, ES and EG cells generate teratomas,
which again demonstrates their multipotency. ES (and EG) cells can be
identified by positive staining with the antibodies SSEA1 and SSEA4.

[0384]At the molecular level, ES and EG cells express a number of
transcription factors highly specific for these undifferentiated cells.
These include oct-4 and Rex-1. Also found are the LIF-R and the
transcription factors sox-2 and Rox-1, even though the latter two are
also expressed in non-ES cells. Oct-4 is a transcription factor expressed
in the pregastrulation embryo, early cleavage stage embryo, cells of the
inner cell mass of the blastocyst, and in embryonic carcinoma (EC) cells.
Oct-4 is down-regulated when cells are induced to differentiate in vitro
and in the adult animal oct-4 is only found in germ cells. Several
studies have shown that oct-4 is required for maintaining the
undifferentiated phenotype of ES cells, and plays a major role in
determining early steps in embryogenesis and differentiation. oct-4, in
combination with Rox-1, causes transcriptional activation of the
Zn-finger protein Rex-1, and is also required for maintaining ES in an
undifferentiated state. Likewise, sox-2, is needed together with oct-4 to
retain the undifferentiated state of ES/EC and to maintain murine (but
not human) ES cells. Human or murine primordial germ cells require
presence of LIF. Another hallmark of ES cells is presence of telomerase,
which provides these cells with an unlimited self-renewal potential in
vitro.

[0385]Stem cells have been identified in most organ tissues. The best
characterized is the hematopoietic stem cell. This is a mesoderm-derived
cell that has been purified based on cell surface markers and functional
characteristics. The hematopoietic stem cell, isolated from bone marrow,
blood, cord blood, fetal liver and yolk sac, is the progenitor cell tint
reinitiates hematopoiesis for the life of a recipient and generates
multiple hematopoietic lineages (see Fei, R., et al., U.S. Pat. No.
5,635,387; McGlave, et al., U.S. Pat. No. 5,460,964; Simmons, P., et al.,
U.S. Pat. No. 5,677,136; Tsukamoto, et al., U.S. Pat. No. 5,750,397;
Schwartz, et al., U.S. Pat. No. 759,793; DiGuisto, et al., U.S. Pat. No.
5,681,599; Tsukamoto, et al., U.S. Pat. No. 5,716,827; Hill, B., et al.,
Exp. Hematol. (1996) 24 (8): 936 943). When transplanted into lethally
irradiated animals or humans, hematopoietic stem cells can repopulate the
erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hemopoietic
cell pool. In vitro, hemopoietic stem cells can be induced to undergo at
least some self-renewing cell divisions and can be induced to
differentiate to the same lineages as is seen in vivo. Therefore, dais
cell fulfills the criteria of a stem cell. Stem cells which differentiate
only to form cells of hematopoietic lineage, however, are unable to
provide a source of cells for repair of other damaged tissues, for
example, heart or lung tissue damaged by high-dose chemotherapeutic
agents.

[0386]A second stem cell that has been studied extensively is the neural
stem cell (Gage F H: Science 287:1433 1438, 2000; Svendsen C N et al,
Brain Path 9:499 513, 1999; Okabe S et al, Mech Dev 59:89 102, 1996).
Neural stem cells were initially identified in the subventricular zone
and the olfactory bulb of fetal brain. Until recently, it vas believed
that the adult brain no longer contained cells with stem cell potential.
However, several studies in rodents, and more recently also non-human
primates and humans, have shown that stem cells continue to be present in
adult brain. These stem cells can proliferate in vivo and continuously
regenerate at least some neuronal cells in vivo. When cultured ex vivo,
neural stem cells can be induced to proliferate, as well as to
differentiate into different types of neurons and glial cells. Widen
transplanted into the brain, neural stem cells can engraft and generate
neural cells and glial cells. Therefore, this cell too fulfills the
definition of a stem cell.

[0389]Compared with ES cells, tissue specific stem cells have less
self-renewal ability and, although they differentiate into multiple
lineages, they are not multipotent. No studies have addressed whether
tissue specific cells express markers described above of ES cells. In
addition, the degree of telomerase activity in tissue specific stem cells
has not been fully explored, in part because large numbers of highly
enriched populations of these cells are difficult to obtain.

[0390]Until recently, it was thought that organ specific stem cells could
only differentiate into cells of the same tissue. A number of recent
publications have suggested that adult organ specific stem cells may be
capable of differentiating into cells of different tissues. A number of
studies have shown that cells transplanted at the time of a bone marrow
transplant can differentiate into skeletal muscle (Ferrari Science
279:528 30, 1998; Gussoni Nature 401:390 4, 1999). This could be
considered within the realm of possible differentiation potential of
mesenchynmal cells that are present in marrow. Jackson published that
muscle satellite cells can differentiate into hemopoietic cells, again a
switch in phenotype within the splanchnic mesoderm Jackson PNAS USA
96:14482 6, 1999). Other studies have shown that stem cells from one
embryonal layer (for instance splanchnic mesoderm) can differentiate into
tissues thought to be derived during embryogenesis from a different
embryonal layer. For instance, endothel cells or their precursors
detected in humans or animals that underwent marrow transplantation are
at least in part derived from the marrow donor (Takahashi, Nat Med 5:434
B, 1999; Lin, Clin Invest 105:71 7, 2000). Thus, visceral mesoderm and
not splanchnic mesoderm, such as MSC, derived progeny are transferred
with the infused marrow. Even more surprising are the reports
demonstrating both in rodents and humans that hepatic epithelial cells
and biliary duct epithelial cells are derived from the donor marrow
(Petersen, Science 284:1168 1170, 1999; Theise, Hepatology 31:235 40,
2000; Theise, Hepatology 32:11 6, 2000). Likewise, three groups have
shown that neural stem cells can differentiate into hemopoietic cells.
Finally, Clarke et al. reported that neural stem cells injected into
blastocysts can contribute to all tissues of the chimeric mouse (Clarke,
Science 288:1660 3, 2000).

[0391]Transplantation of tissues and organs generated from heterologous
embryonic stem cells requires either that the cells be further
genetically modified to inhibit expression of certain cell surface
markers, or that the use of chemotherapeutic immune suppressors continue
in order to protect against transplant rejection. Thus, although
embryonic stem cell research provides a promising alternative solution to
the problem of a limited suppler of organs for transplantation, the
problems and risks associated with the need for immunosuppression to
sustain transplantation of heterologous cells or tissue would remain. An
estimated 20 immunologically different lines of embryonic stem cells
would need to be established in order to provide immunocompatible cells
for therapies directed to the majority, of the population (Wadman, M.,
Nature (1999) 398: 551). Using cells from the developed individual,
rather than an embryo, as a source of autologous or allogeneic stem cells
would overcome the problem of tissue incompatibility associated with the
use of transplanted embryonic stem cells, as well as solve the ethical
dilemma associated with embryonic stem cell research.

[0392]A method for differentiating mammalian stem cells according to one
embodiment can include (a) preparing a medium containing mammalian stem
cells and placing the medium in a system according embodiments, (b)
adding to the medium an effective amount of an agent which causes
differentiation of the cells, producing differentiated cells, (c)
contacting the cells from step (b) with an effective amount of an agent
that causes stabilization of cells produced in step (b) and (d)
recovering stabilized, differentiated cells.

[0393]A method for generating differentiated cells from mammalian
mesenchymal stem cells according to one embodiment can include (a)
placing the mesenchymal stem cells in a system according embodiments, (b)
incubating the mesenchymal stem cells under conditions that induce the
mesenchymal stem cells to differentiate and (c) recovering the
differentiated cells.

[0394]A method of producing a genetically engineered cell such as stem
cells according to one embodiment can include (a) placing cells such as
stem cells in a system according embodiments under conditions that do not
cause the cells to differentiate, (b) transfecting the cells such as stem
cells with a DNA construct including at least one gene of interest, (c)
selecting for expression of the gene of interest in the cells such as
stem cells and (d) culturing the cells such as stem cells selected in
step (c).

[0395]A method of in vivo administration of a protein or gene of interest
according to one embodiment can include (a) placing cells such as stem
cells in a system according embodiments, (b) transfecting the cells such
as stem cells with a vector including DNA or RNA that expresses a protein
or gene of interest, (c) selecting for expression of the protein or gene
of interest in the cells such as stem cells and (d) delivering the cells
such as stem cells selected in step (c) to a mammal in need thereof.

[0396]A method of testing the ability of a candidate agent to modulate the
proliferation of a lineage uncommitted cell according to one embodiment
can include (a) placing stem cells in a system according embodiments, (b)
culturing the stem cells in a growth medium that maintains the stem cells
as lineage uncommitted cells, (c) adding the candidate agent to the
medium and (d) determining the proliferation and lineage of the cells by
mRNA expression, antigen expression or other means.

[0397]A method of preparing a stem cell matrix for use in tissue or organ
repair according to one embodiment can include (a) admixing a preparation
including stem cells with a physiologically acceptable matrix material to
form a stem cell matrix and (b) incubating the stem cell matrix in a
system according embodiments prior to use in tissue or organ repair or
treatment.

[0398]A method of tissue or organ repair or treatment, according to one
embodiment can include (a) preparing a stem cell matrix by admixing a
preparation including stem cells with a physiologically acceptable
matrix, (b) incubating the stem cell matrix in a system according to
embodiments prior to use and (c) introducing the stem cell matrix into a
patient in need thereof.

[0399]In accordance with another embodiment, mammalian stem cells can be
pluripotent or multipotent stem cells or totipotent stem cells. In
accordance with another embodiment, stem cells can be homogeneous stem
cells or heterogeneous stem cells. In accordance with another embodiment,
stem cells can be autologous or allogeneic to a recipient or a mammal.

[0401]In accordance with another embodiment, stem cells can be obtained
from a tissue selected from the group consisting of adult, embryonic, and
fetal tissue. In accordance with another embodiment, such tissue can
include bone marrow, muscle, adipose, liver, heart, lung, or nervous
system tissue.

[0403]In accordance with another embodiment, stem cells are affixed to a
physiologically acceptable matrix material using a biological adhesive
such as fibrin glue. In accordance with another embodiment, the fibrin
glue can be supplemented with at least one agent. In accordance with
another embodiment, a physiologically acceptable matrix material can be
absorbable or non-absorbable.

[0404]A method for producing a protein of interest according to one
embodiment can include (a) culturing a host cell in a system according to
disclosed embodiments under conditions in which the protein is expressed;
and (b) recovering the protein. A method for maintaining a culture of
cells according to one embodiment can include placing the cells in a
system according to disclosed embodiments under conditions in which the
cells remain substantially unchanged for an extended period of time. In
accordance with another embodiment, an isolated culture of cells can be
prepared according to disclosed embodiments of methods.

[0405]In accordance with another embodiment, immune cells (e.g., killer
cells, T-cells or the like) can be used in various fluids as described
herein or used in systems 1, 1101. Further, immune cells can be used in
various fluids during preparation or differentiation of stem cells
according to methods otherwise known in the art. Such immune cells can be
used to clean or decontaminate contaminated cells. Further, such cells
can be used to detect otherwise undetectable contaminants such as fungi
or mold. In addition, according to embodiments, the immune cells can
immunize or destroy contaminants such as mycoplasma, fungi, bacteria or
the like. Accordingly, the effectiveness of biological embodiments
disclosed herein can be increased.

[0406]Embodiments of the system may be applied at any or all the stages of
cardiovascular disease, such as the early to late stages and spanning
through drug treatment to tissue and cell regeneration. The early stages
are often treated medically with drugs and biomolecules that can be
screened and tested, discovered and developed using the technology. The
next stages are often treated with drug-eluting stents where in this
example, the `drug-elutant` can be discovered, screened, and tested,
discovered and developed using embodiments of the system. The late stages
often require arterial bypass where embodiments of the system can be used
to produce tissue regenerated or engineered products, for example
arteries, from patient cells such as, for example, stem cells or
progenitor cells with various possible scaffolds such as a ePTFE or
collagen composites.

[0407]Embodiments of the system may also be used as a vascular trainer, to
recondition a vein or artery under various dynamic conditions, with
various growth factors or genetic or chemical treatment and other
additives enhancing the therapeutic outcome of such a conditioning
environment. This may also be also useful in reviving cryogenically
preserved specimens.

[0408]The invention also provides embodiments of a system and a method by
which appropriate mechanical environments are applied ex vivo to direct
the remodeling of small, excised blood vessels to create
tissue-engineered vessels characterized by increased length, internal
diameter, and wall thickness. Thus, the small excised vessels, arteries,
or even veins, become tissue-engineered blood vessels for use in vascular
surgery. Embodiments of the invention further provide an evaluation of
the performance of these tissue-engineered blood vessels in vivo.

[0409]Embodiments of the system allow investigations of the hypothesis
that longitudinal stress or strain induces artery elongation. In
addition, while there are autologous donor arteries with proper diameter
and wall thickness for vascular grafts, they often are of an insufficient
length to meet the required need. For example, the internal thoracic
artery has excellent long-term patency, but is of an adequate length for
only a single bypass graft. However, recognizing that if the artery could
be elongated, it could be used to bypass multiple occlusions, and the use
of vessels demonstrating inferior performance could be avoided,
embodiments of the invention advantageously provides reliable
tissue-engineered blood vessels of sufficient length to meet his need.

[0410]In addition, embodiments of the system are further used to explore
the molecular regulation of mechanically induced vascular remodeling by
characterizing the expression and regulation of key regulatory factors,
for which the spatial expression and distribution of mRNA and protein are
monitored as a result of various mechanical loads.

[0411]Thus, embodiments of the invention also provide a protocol by which
localized intravascular and extravascular pressures are measured in real
time, and the measured pressures are compared with the calculated
pressure estimates.

[0412]The graft tissue component of the vascular graft may be derived from
essentially any biological tissue of interest provided the tissue has the
proper geometrical dimensions and/or configurations for its intended
application. Typically, the graft tissue will be comprised of vascular
tissue removed from a human or from an animal species, e.g., bovine,
porcine, ovine, equine, canine, goat, etc., and may be removed from
various anatomical positions within the body. For example, the graft
tissue may be derived from carotid arteries, thoracic arteries, mammary
arteries, and the like. The graft tissue must have a structure, e.g., a
tubular structure, which defines an interior lumen having dimensions
sufficient for allowing blood to flow therethrough following
implantation.

[0413]The primary component of the biological tissues used to fabricate
bioprostheses is collagen, a generic term for a family of related
extracellular proteins. Collagen molecules consists of three chains of
poly (amino acids) arranged in a trihelical configuration ending in
non-helical carboxyl and amino termini. These collagen molecules assemble
to form microfibrils, which in turn assemble into fibrils, resulting in
collagen fibers. The amino acids which make up the collagen molecules
contain side groups, including amine, acid and hydroxyl groups, in
addition to the amide bonds of the polymer backbone, all of which are
sites for potential chemical reaction on these molecules.

[0414]Because collagenous tissues degrade very rapidly upon implantation,
it is preferable to stabilize the tissue if it is to be implanted into a
living system. The tissue can be stabilized using embodiments of the
system of the invention in combination with any of a variety of
conventional approaches. For example, chemical stabilization by tissue
cross-linking, also referred to as tissue fixation, can be achieved using
bi-functional and multi-functional molecules having reactive groups
capable of forming irreversible and stable intramolecular and
intermolecular chemical bonds with the reactive amino acid side groups
present on the collagen molecules. An additional method for the
fixation/stabilization of the graft tissues involves a photooxidation
process.

[0415]Such photooxidation may be carried out according to conventional
methodologies. Suitable photooxidation process have been described, for
example in U.S. Pat. No. 5,854,397, the disclosure of which is
incorporated herein by reference, and in Moore et al. (1994). The
photooxidation process provides an efficient and effective method for
cross-linking and stabilizing various proteinaceous materials including,
but not limited to, collagen, collagen fibrils and collagen matrices. The
term proteinaceous material as used herein includes both proteins such as
collagen and protein-containing materials such as tissues. The material
to be cross-linked is generally provided as a vascular tissue sample.
Such materials are harvested from the donor animal and immediately
immersed in cold buffered saline for storage, with frequent rinses and/or
changes with fresh saline, until a fixation process is performed.

[0416]The vascular tissue material to be photooxidized is then immersed,
dispersed, or suspended (depending upon its previous processing) in an
aqueous media for processing. Suitable media for immersion of the
material (for purposes of convenience, the word "immersion" shaft be
considered to include suspension and/or solubilization of the
proteinaceous material) include aqueous and organic buffer solutions
having a neutral to alkaline pH, preferably a pH of about 6.5 and above
because of the denaturation caused by add pH. Particularly preferred are
buffered aqueous solutions having a pH of from about 6.8 to about 8.6.

[0417]In a preferred photooxidation process, two media solutions are
utilized for what is referred to herein as "preconditioning" the vascular
tissue material before irradiation. The material is "preconditioned" in
the sense that tissue soaked in the first media solution and irradiated
in the second are apparently better cross-linked, e.g., they show
improved mechanical properties and decreased susceptibility to
proteolytic degradation. The efficacy of this preconditioning is affected
by the osmolality of the first media solution, it being preferred that
solutions of high osmolality be used as the first media solution.
Particularly preferred are sodium potassium, or organic buffer solutions
such as sodium, chloride, sodium phosphate, potassium chloride, potassium
phosphate, and Good's buffers having a pH of from about 6.8 to about 8.6,
the osmolality of which have been increased by addition of a solute such
as 4M sucrose or other soluble, high molecular weight carbohydrate to
between about 393 mosm and about 800 mosm.

[0418]The solute added to increase the osmolality of the first media may
have an adverse effect on the degree of cross-linking of the product when
present during irradiation. Consequently, after soaking in the first
media, the tissue is preferably removed therefrom and immersed in a
second media for irradiation. The second media is preferably an aqueous
buffered solution having a pH of from about 6.8 to about 8.6 in which the
photo-catalyst is dissolved. Preferred second media are sodium and
potassium phosphate buffers having a pH of from about 7.4 to about 8.0
and an osmolality of from about 150 to about 400 mosm.

[0419]The tissue may be advantageously immersed sequentially in the first
media and then in the catalyst-incorporated second media prior to
photooxidation for a total period of time sufficient to allow tissue,
dye, and medium to reach equilibrium. When the ratio of the concentration
of the medium to that of the material to be cross-linked is in the range
of from about 10:1 to 30:1, equilibrium can generally be readily
achieved. The ratio of the concentrations is generally not critical, and
may be adjusted up or down as desired. Once an equilibrium is reached,
the sample is photooxidized in the catalyst-incorporated medium. The time
required to reach equilibrium varies depending upon such factors as, for
instance, the temperature of the media solutions, the osmolality of the
first media, and the thickness of the tissue or other sample of
proteinaceous material. A period of time as short as a few minutes or as
long as several days may be sufficient, but it has been found that
periods of from minutes to hours duration is generally sufficient to
allow sufficient time for most collagenous materials and media to
equilibrate.

[0420]The catalysts for use in the photofixation process include
photooxidative catalysts (photo-catalysts) that when activated will cause
transfer of electrons or hydrogen atoms and thereby oxidize a substrate
in the presence of oxygen. Although varied results are possible depending
upon the particular catalyst utilized, appropriate catalysts include, but
are not limited to, those listed in Oster, et al., J. Am. Chem. Soc. 81:
5095, 5096 (1959). Particularly preferred catalysts include methylene
blue, methylene green, rose bengal, riboflavin, proflavin, fluorescein,
eosin, and pyridoxal-5-phosphate.

[0421]The concentration of catalyst in the media will vary based on
several process parameters, but should be sufficient to insure adequate
penetration into the material to be cross-linked and to catalyze the
photooxidation of the protein. A typical catalyst concentration ranges
from about 0.0001%-0.25% (wt/vol); the preferred concentration ranges
from about 0.001 to about 0.01%.

[0422]To achieve maximum cross-linking and stabilization of the vascular
tissue, the following steps may be taken: (1) the photooxidative catalyst
should be completely solubilized in the reaction medium prior to use to
ensure that the desired dye concentration is achieved; (2) the
concentration of the catalyst in the tissue or suspension should be in
equilibrium with that in the surrounding medium; and (3) the catalyst
solution should be filtered to remove any sizable particulate matter,
including chemical particulates, therefrom.

[0423]Because the photofixation process involves primarily an oxidative
reaction, to assure completion of the reaction, an adequate supply of
oxygen must be provided during photooxidation. While an oxygen
concentration of about 20% by volume (referring to the concentration of
oxygen in the atmosphere over the media) is preferred to assure
sufficient dissolved oxygen in the media to prevent oxygen content from
becoming rate limiting, all concentrations >0% can also be used.
Depending upon the temperature at which the material is held during
exposure to light, the oxygen requirement can be met, for instance, by
agitating the solution or otherwise mixing the solution, suspension, or
sample during the reaction process. Oxygen concentration in the
atmosphere over the media during irradiation is preferably maintained in
the range of from about 5% to about 40%. Such concentrations (again
depending upon temperature) can also be achieved, for instance, by
bubbling air into the media during irradiation of the tissue or, if
concentrations higher than about 20% are desired, by bubbling oxygen
mixtures or air having an increased oxygen content into the media.

[0424]As with other catalytic or kinetic-type reactions, the temperature
at which the reaction is run directly affects the reaction rate and the
oxygen available in the media. Tests conducted with various media ranging
in pH from about 6.8 up to about 7.4 indicate that as the temperature of
the media increases from about 4 C to about 50 C., oxygen concentration
drops in roughly linear fashion from about 11-12 ppm to about 5 ppm. The
dye-catalyzed photooxidation process is exothermic, and it is, therefore,
preferred that a relatively constant temperature be maintained during
irradiation of the proteinaceous material to prevent denaturation of the
proteinaceous material and the driving of the oxygen out of the media by
the increase in temperature. Usually, a recirculating bath is sufficient
to maintain and control the temperature within the jacketed reaction
vessel or chamber but placement of the reaction chamber within a
controlled environment such as a refrigerator or freezer will work as
well. As disclosed herein, photooxidation conducted at temperatures
ranging from about -2 C to 40 C. has been shown to be effective; the
preferred temperatures being about 0 C to about 25 C. To prevent or
alleviate denaturation of the protein comprising the vascular tissue,
temperatures below the denaturation temperature of that protein are
preferred. Likewise, temperatures above the freezing point of the
reaction medium are also preferred.

[0425]The process is conducted at temperatures low enough to avoid heat
denaturation and pH high enough to avoid acid denaturation of the
collagen or other proteinaceous material during cross-linking. Likewise,
temperature is held at a level sufficient to maintain the oxygen
concentration in the media in which the proteinaceous material is
immersed during irradiation.

[0426]Once the tissue is prepared, it is photo-irradiated, preferably in a
controlled system wherein temperature, distance to light source,
irradiation energy and wavelength, oxygen concentration and period of
irradiation can be monitored and/or maintained. The tissue is
photo-irradiated under conditions sufficient to cause cross-linking.
Photooxidation is generally achieved using incandescent, white light or
fluorescent light, i.e., visible light, or that portion of light in the
visible range that is absorbed by the catalyst.

[0427]The intensity of the light employed, and the length of time required
to cross-link a given proteinaceous material will vary depending upon
several factors. These include: (1) the type and amount of proteinaceous
material; (2) the thickness of the tissue sample; (3) the distance
between the proteinaceous material and the irradiation source; (4) the
catalyst employed; (5) the concentration of catalyst; and (6) the type
and intensity of the light source. For instance, exposure time may vary
from as little as a few seconds up to as much as about 160 hours. With
regard to the intensity of the light, one or more lights may be used of
intensity preferably ranging up to about 150 watts, preferably held at a
distance from about 2.5 cm to 12 cm from the sample surface. Greater
exposure time is required when fluorescent or lower power lights are
utilized. These ranges are quite variable; however, they may be easily
determined for a given material without resort to undue experimentation.

[0428]Evidence of the cross-linking of the vascular tissue by
photooxidation may be provided by several approaches. For instance,
polyacrylamide gel electrophoresis of the irradiated material in sodium
dodecylsulfate (for example, 0.1%) evidences such cross-linking by a
significant decrease in the amount of lower molecular weight material
with the simultaneous appearance of high molecular weight material.

[0429]Further evidence of cross-linking may be provided by known
solubility and digestibility tests. For instance, cross-linked collagen
is generally insoluble such that solubility tests provide direct evidence
of the degree of cross-linking. The digestibility tests involve
incubation of the proteinaceous product with a proteolytic enzyme such as
papain, trypsin, pepsin, or bacterial collagenase, and the subsequent
testing of the media in which the product and enzyme are incubated for
soluble degradation products of the cross-linked product. The test is
generally accomplished by pelletizing the undigested, cross-linked tissue
by centrifugation and testing the resulting supernatant for degradation
products.

[0430]Following photo-irradiation, the cross-linked product may be
advantageously subjected to additional treatments for the removal of the
catalyst and other chemicals or impurities found therein before being
used as a vascular graft. Multiple rinses in a fresh buffer solution, for
example, may be used, followed by at least partial removal of water by
treatment with, for instance, ethanol. The number of rinses and the
volume of rinse solution required depend upon the mass of the tissue and
the catalyst concentration utilized.

[0431]In addition to the use of photooxidation processes for the fixation
of the graft tissue, numerous other fixation methods have been described
and are readily available in the art and may be used in conjunction with
embodiments of the invention. For example, glutaraldehyde, and other
related aldehydes, have seen widespread use in preparing cross-linked
biological tissues. Methods for glutaraldehyde fixation of biological
tissues have been extensively described and are well known in the art. In
general, a tissue sample to be cross-linked is simply contacted with a
glutaraldehyde solution for a duration effective to cause the desired
degree of cross-linking within the biological tissue being treated.

[0432]Many variations and conditions have been applied to optimize
glutaraldehyde fixation procedures. For example, lower concentrations
have been found to be better in bulks tissue cross-linking compared to
higher concentrations. It has been proposed that higher concentrations of
glutaraldehyde may promote rapid surface cross-linking of the tissue,
generating a barrier that impedes or prevents the further diffusion of
glutaraldehyde into the tissue bulk. For most bioprosthesis applications,
the tissue is treated with a relatively low concentration glutaraldehyde
solution, e.g., typically between 0.1%-5%, for 24 hours or more to ensure
optimum fixation. Of course, various other combinations of glutaraldehyde
concentrations and treatment times will also be suitable depending on the
objectives for a given application.

[0433]In addition to bifunctional aldehydes, many other chemical fixation
procedures have been described (for review, see Khor, Biomaterials 18:
95-105, 1997). For example, some such methods have employed polyethers,
polyepoxy compounds, diisocyanates, azides, etc. These and other
approaches available to the skilled individual in the art for treating
biological tissues wills be suitable for cross-linking vascular graft
tissue in embodiments of systems according to his invention.

[0434]The hemodynamic forces recreated within and by embodiments of the
system may also be used to improve organ transplant procedures and make
these procedures more successful by providing an appropriate environments
(e.g., hemodynamic) for an organ prior to transplant, both during the
transport period and while awaiting actual transplant. More particularly,
providing a simulated pulsatile or hemodynamic environment, which may
represent in vivo conditions of the particular organ, to the organ during
these periods protects the integrity of the organ by maintaining its
proper functionality after it has been removed so as to provide the best
possible transition and adaptation in a new host. Also, embodiments of
the system may be used to re-vive an organ that was cryopreserved or
treated with a type of preservation treatment as well.

[0435]The invention also provides an embodiment of a system for applying
controlled shear flow stress to mammalian cell cultures used for
artificial cartilage production.

[0436]Applying shear flow stress to a three-dimensional or monolayer
chondrocyte culture advantageously increases the ratio of type II to type
I collagen produced by the chondrocytes. The shear flow stress also
advantageously enhances maintenance of the chondrocyte phenotype. Thus,
application of shear flow stress according to embodiments of this
invention improves the functional outcome of a three-dimensional or
monolayer chondrocyte culture and increases the useful lifetime of the
monolayer culture.

[0437]Applying shear flow stress to stem cells induces or promotes
differentiation of the stem cells into chondrocytes. Inducing or
promoting stem cells to differentiate into chondrocytes is accomplished
by substituting stem cells for chondrocytes in the shear flow method
described herein with regard to chondrocytes. The chondrocytes arising
from the stem cell differentiation process are maintained in the culture,
under shear flow stress, for a sufficient time to allow production of
artificial cartilage.

[0438]Shear flow stress also can be used according to embodiments of this
invention to induce transdifferentiation of differentiated cells into
chondrocytes. Transdifferentiation is accomplished by substituting,
differentiated cells other than chondrocytes, e.g., myoblasts or
fibroblasts, in the shear flow method described herein with regard to
chondrocytes. In response to the shear flow stress, the differentiated
cells transdifferentiate into chondrocytes. The chondrocytes arising from
the transdifferentiation process are maintained in the culture, under
shear flow stress, for a sufficient time to allow production of
artificial cartilage.

[0439]Artificial cartilage produced according to any embodiment of this
invention can be used for surgical transplantation, according to
established medical procedures, to replace damaged or missing cartilage.
Typically, artificial cartilage is employed in the repair of human
joints, e.g., knees and elbows.

[0440]Preferably, the cultured chondrocytes are anchored, i.e., attached,
to a substrate, whether grown as a monolayer or grown in a 3-dimensional
culture. A monolayer-supporting surface, or a 3-dimensional scaffold, in
a bioreactor is inoculated with chondrocytes, stem cells, or
differentiated cells suitable for transdifferentiation. Artificial
cartilage can be produced by growing chondrocytes in a conventional
mammalian tissue culture medium, e.g., RPMI 1640, Fisher's, Iscove's or
McCoy's. Such media are well known in the art, and are commercially
available. Typically, the cells are cultured at 37 C in air supplemented
with 5% CO2. Under these conditions, a chondrocyte monolayer or a three
dimensional cartilage matrix is produced in approximately 7 to 56 days,
depending on the cell type used for inoculation and the culture
conditions.

[0441]Isolated chondrocytes can be used to inoculate the surface of a
support or a 3-dimensional matrix. Alternately, stem cells, or cells
suitable for transdifferentiation can be used for inoculation.

[0442]Cells used for inoculation of cultures used in the invention can be
isolated by any suitable method. Various starting materials and methods
for chondrocyte isolation are known. See generally, Freshney, Culture of
Animal Cells. A Manual of Basic Techniques, 2d ed., A. R. Liss Inc., New
York, pp 137-168 (1987). Examples of starting materials for chondrocyte
isolation include mammalian knee joints or rib cages.

[0443]If the starting material is a tissue in which chondrocytes are
essentially the only cell type present, e.g., articular cartilage, the
cells can be obtained directly by conventional collagenase digestion and
tissue culture methods. Alternatively, the cells can be isolated from
other cell types present in the starting material. One known method for
chondrocyte isolation includes differential adhesion to plastic tissue
culture vessels. In a second method, antibodies that bind to chondrocyte
cell surface markers can be coated on tissue culture plates and then used
to selectively bind chondrocytes from a heterogeneous cell population. In
a third method, fluorescence activated cell sorting (FACS) using
chondrocyte-specific antibodies is used to isolate chondrocytes. In a
fourth method, chondrocytes are isolated on the basis of their buoyant
density, by centrifugation through a density gradient such as Ficoll.

[0444]Examples of tissues from which stem cells for differentiation, or
differentiated cells suitable for transdifferentiation, can be isolated
include placenta, umbilical cord, bone marrow, skin, muscle, periosteum,
or perichondrium. Cells can be isolated from these tissues by explant
culture and/or enzymatic digestion of surrounding matrix using
conventional methods.

[0445]When the artificial cartilage construct has grown to the desired
size and composition, a cryopreservative fluid can be introduced into
embodiments of the system. The cryopreservative fluid freezes the
artificial cartilage construct for future use. Cryopreservation methods
and materials for mammalian tissue culture material are known to those of
ordinary skill in the art.

[0446]Methods and materials for 3-dimensional cultures of mammalian cells
are known in the art. See, e.g., U.S. Pat. No. 5,266,480. Typically, a
scaffold is used in a bioreactor growth chamber to support a
3-dimensional culture. The scaffold can be made of any porous, tissue
culture-compatible material into which cultured mammalian cells can enter
and attach or anchor. Such materials include nylon (polyamides), dacron
(polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl
chloride, polytetrafluoroethylene (teflon), nitrocellulose, and cotton.
Preferably, the scaffold is a bioabsorbable or biodegradable material
such as polyglycolic acid, catgut suture material, or gelatin. In
general, the shape of the scaffold is not critical.

[0447]Optionally, prior to inoculating chondrocytes into the scaffold,
stromal cells are inoculated into the scaffold and allowed to form a
stromal matrix. The chondrocytes are then inoculated into the stromal
matrix. The stromal cells can include fibroblasts. The stromal cells can
also include other cell types.

[0448]A 3-dimensional culture can be used in a system of the invention and
shear flow stress applied to the chondrocytes by the movement of the
liquid culture medium pumped through the growth chamber, which contains
the 3-dimensional culture. Preferably, in such embodiments, the scaffold
and attached cells are static.

[0449]Embodiments of the system for simulating hemodynamic forces as
embodied and broadly described herein is capable of generating the
complete range of hemodynamic force patterns in the interest and
advancement of cardiovascular research, and will make new avenues of
research and development available which were never before possible, at
any cost. Embodiments of systems and methods will greatly advance our
understanding of cardiovascular function and disease, and allow
pharmacologic and genetic strategies to be tested at much lower costs
than conventional methods of experimentation. Ultimately, patients will
benefit the most, since embodiments of the invention will advance new
concepts in cardiovascular disease progression, development, and
treatment. Healthy patients can function as productive members of
society, improve their quality of life, and reduce the cost of medical
treatment.

[0450]Hemodynamic conditions are one class of dynamic conditions (FIG. 18)
and affect cardiovascular physiology and pathology. Pulsatile flow (Q),
pressure (P), and diameter (D) waveforms exert wall shear stress (WSS),
normal stress, and circumferential strain (CS) (types of dynamic
conditions as shown in FIG. 17) on blood vessels. In vitro studies to
date have focused on either WSS or CS but not their interaction. Studies
caused at using embodiments of systems 1 and 1101 have demonstrated that
concomitant WSS and CS affect endothelial cell (EC) biochemical response
modulated by the temporal phase angle between WSS and CS (stress phase
angle, SPA) (one type of dynamic condition as shown in FIG. 17). Systems
1, 1101 have shown that large negative SPA occurs in regions of the
circulation where atherosclerosis and intimal hyperplasia are prevalent,
and that nitric oxide (NO) biochemical secretion was significantly
decreased in response to a large negative SPA of -180 deg with respect to
an SPA of 0° in bovine aortic endothelial cells (BAEC) at 5 hr.
Systems 1, 1101 use the discrete hemodynamic conditions of
pro-atherogenic (SPA=-180 deg) and normopathic (SPA=0 deg) states as
input information to study the physiologic SPA used to produce the
corresponding hemodynamic conditions at the tubular structures.
Accordingly, systems 1, 1101 demonstrate that one type of dynamic
condition (SPA) plays an important role in hemodynamics with respect to
vascular remodeling, homeostasis, and pathogenesis, and that a large
negative SPA is pro-atherogenic.

[0455]Nitric oxide (NO) is one of the smallest biomolecules produced in
mammalian cells and plays a major role in vascular homeostasis, as
discussed, for example, in Ignarro, L. J., 1990, "Nitric Oxide. A Novel
Signal Transduction Mechanism for Transcellular Communication,"
Hypertension, 16(5), pp. 477-483, the contents of which are incorporated
herein by reference. The content longitudinal and/or radial velocity/flow
concentration of NO are types of dynamic conditions FIG. 17). The small
size of NO permits unhindered movement to neighboring cells, however, the
short half-life (<5 seconds) limits its range. Red blood cells can aid
in the transport of NO through binding with hemoglobin to form
nitrosyl-heme adducts that are more stable than free NO. NO production
occurs trough a redox reaction involving three cosubstrates, five
cofactors, and nitric oxide synthase (NOS) that leads to the conversion
of L-arginine to L-citruline and release of NO. See, for example, Nathan,
C., and Xie, Q. W., 1994, "Nitric Oxide Synthases: Roles, Tolls, and
Controls," Cell, 78(6), pp. 915-918 and Nathan, C., and Xie, Q. W., 1994,
"Regulation of Biosynthesis of Nitric Oxide," J. Biol. Chem., 269(19),
pp. 13725-13728, the contents of which are incorporated herein by
reference.

[0457]Pulsatile blood flow in the arterial circulation produces
oscillatory wall shear stress with mean values from 5 to 40
dyne/cm2. See, for example, Lipowsky, H. H., 1995, "Shear Stress in
the Circulation," in Flow-dependent Regulation of Vascular Function,
edited by J. A. Bevan et al., the contents of which are incorporated
herein by reference. Pulsatile blood pressure causes large arteries to
expand predominantly in the circumferential direction, whereas
longitudinal expansion is constrained by blood vessel branching and
tethering. See, for example, Dobrin, P. B., 1978, "Mechanical Properties
of Arteries," Physiol. Rev., 58, pp. 397-460, the contents of which are
incorporated herein by reference. As the vessel expands, a uniform
circumferential strain is produced. For this reason, a three-dimensional
geometry tube or tubular structure, instead of a two-dimensional flat
membrane, is used in systems 1, 1101, which produces heterogeneous strain
fields. See, for example, Brown, T. D., 2000, "Techniques for Mechanical
Stimulation of Cells in Vitro: A Review," J. Biomech., 33, pp. 3-14, the
contents of which are incorporated herein by reference. In one embodiment
of the invention, systems 1, 101, 1101 produce a maximum cyclic strain or
diameter variation, CS=(Dmax-Dmin)/Dmean, driven by
pulsing transmural pressure in large arteries such as the thoracic aorta,
carotid artery, femoral artery, and pulmonary artery ranges from 2% to
18% over the pressure pulse. The venous systemic circulation has almost
no diameter variation due to the low pressure pulse. Atherosclerosis
occurs in the large arteries where CS is significant. Accordingly in one
embodiment of the invention, systems 1, 1101 produces both hemodynamic
conditions CS and WSS.

[0458]Blood vessel endothelial cells in vivo are subjected to simultaneous
pulsatile CS and WSS acting approximately in perpendicular directions.
The temporal phase angle between pressure and flow (e.g., impedance phase
angle, IPA also a type of dynamic condition as per FIG. 17) generated by
global wave reflection in the circulation, as well as the inertial
effects of blood flow, cause temporal phase shifts to occur between CS
and WSS. The temporal phase angle between CS and WSS (SPA) in vivo
generates complex, time-varying mechanical force patterns on the EC
monolayer, as shown in FIGS. 10A-10H, 25A-25C and 30-34.

[0459]Physiologic factors contribute to variations in SPA throughout the
circulation. SPA can be described as the phase angle between diameter (D)
and WSS (τ), denoted as φ(D-τ), that shows CS is generally
synchronous with vessel diameter D) variation. The SPA can be decomposed
into two parts

(D-τ)=φ(D-Q)-φ(τ-Q)≈φ(P-Q)-φ(τ-Q)

where φ(D-Q) is approximately equal to the IPA, φ(P-Q) since
diameter (D) and pressure (P) are nearly in phase for an elastic vessel
or tubular structure, and φ(τ-Q) is the phase angle between the
WSS and flow rate. φ(P-Q) is determined from distal resistance,
compliance, and wave reflections. φ(P-Q) of the first harmonic of a
physiologic waveform approaches -45 deg (P lags Q by 45 deg) in the aorta
and large arteries that feed high impedance flow circuits (except for
coronary arteries due to their unique flow circuit), approaches 0 deg in
small arteries due to reduced distal compliance, and also approaches 0
deg in veins that feed low impedance flow circuits. See, for example,
Nichols, W. W., and O'Rourke, M. F., 1998, McDonald's Blood Flow in
Arteries Theoretical, Experimental, and Clinical Principles, Arnold and
Oxford University Press, New York, the contents of which are incorporated
herein by reference.

[0460](τ-Q), the shear-flow phase angle, in straight vessels is
determined by the relative importance of unsteady inertia and viscous
forces and depends strongly on the unsteadiness parameter
[α≡α {square root over ((w/ν))}; α=vessel
radius, w=fundamental frequency of the heart beat, and v=kinematic
viscosity of blood. For large straight arteries and veins with high
α,φ(τ-Q) approaches +45 deg and for small, straight
arteries and veins with low α,φ(τ-Q) approaches 0 deg,
which systems 1, 1101 can produce in specimen 12 or tubular structure
1112. See, for example, Womersley, J. R., 1955, "Method for Calculation
of Velocity) Rate of Flow and Viscous Drag in Arteries When the Pressure
Gradlent is Known," J. Physiol. (London), 127, pp. 553-563, the contents
of which are incorporated herein by reference.

[0461]Based on the above discussion, the following SPA approximations in
straight vessels can be summarized and produced by systems 1, 1101:

Large artery (straight): φ(D-τ)=-45 deg-45 deg=-90 deg

Large vein (straight): φ(D-τ)=0 deg-45 deg=-45 deg

Small artery (straight): φ(D-τ)=0 deg-0 deg=0 deg

Small vein (straight): φ(D-τ)=0 deg-0 deg=0 deg

[0462]The shear-flow phase angle is strongly dependent on local vessel
geometric factors that can induce spatial skewing of velocity profiles
and flow separation. This can lead to local spatial distribution of SPA
(a type of dynamic condition as shown in FIG. 17) in certain vessels such
as those associated with intimal hyperplasia and atherosclerosis. Several
high-risk arterial geometries include the aortic abdominal bifurcation
(see, for example, Lee, C. S., and Tarbell, J. M., 1997, "Wall Shear Rate
Distribution in an Abdominal Aortic Bifurcation Model: Effects of Vessel
Compliance and Phase Angle Between Pressure and Flow Waveforms," J.
Biomech. Eng., 119(3), pp. 333-342, the contents of which are
incorporated herein by reference) curved coronary artery (see, for
example, Qiu, Y., and Tarbell, J. M., 2000, "Numerical Simulation of
Pulsatile Flow in a Compliant Curved Tube Model of a Coronary Artery," J.
Biomech. Eng., 122(1), pp. 77-85, the contents of which are incorporated
herein by reference), and end-to-end undersized graft anastomosis, all of
which can be produced at a specimen 12 in systems 1, 101, 1101. For
example, in the aortic abdominal bifurcation, the SPA drops along the
outer wall, especially near the disease-prone region opposite the flow
divider to -80 deg (e.g., normal) and -100 deg (e.g., hypertensive case).
This region of complex hemodynamic conditions in the aortic abdominal
bifurcation is also characterized by low shear stress as opposed to the
high shear region of flow divider. The inner wall (flow divider) has a
higher SPA (e.g., -20 deg normal and -55 deg hypertensive) and higher
shear stress. Systems 1, 1101 can produce all of these hemodynamic
conditions at specimen 12 or tubular structure 1112. Not only is the SPA
large and negative in the region of atherosclerotic plaque development,
but also hypertension will further decrease the SPA, resulting in a more
atherogenic condition. Such pathology (e.g., dynamic conditions as shown
in FIG. 17) can be reproduced by systems 1, 1101.

[0463]A curved coronary artery experiences complex hemodynamics primarily
caused by the unique coronary flow circuit that allows for the most
extreme SPA in the cardiovascular circulation. The entire coronary artery
experiences a large negative SPA (e.g., SPA<-180 deg: -250 deg on the
inner wall, -220 deg on the outer wall) which can be produced at specimen
12 or tubular structure 1121 by systems 1, 1101. Coronary arteries are
the most disease-prone arteries in the cardiovascular circulation. In all
instances, the SPA is more negative in low shear pathologic regions than
in high shear healthy regions.

[0464]Thus, regions of the circulation prone to pathologic development
such as atherosclerosis and intimal hyperplasia are characterized by
large negative SPA values relative to regions typically without
pathologic development (e.g., veins, small arteries, high shear regions
in large arteries). Accordingly, pathologic development or conditions can
be modeled using systems 1, 1101. Endothelial biomolecule production is
affected by a negative SPA (-100 deg). See, for example, Qiu, Y., and
Tarbell, J. M., 2000, "Interaction Between Wall Shear Stress and
Circumferential Strain Affects Endothelial Cell Biochemical Production,"
J. Vasc. Res., 37(3), pp. 147-157, the contents of which are incorporated
herein by reference.

[0465]Detection of fluid molecules (e.g., endothelial cell NO production)
can demonstrate affects of dynamic conditions of FIG. 17 (e.g., highly
negative SPA) for a class of dynamic conditions, as shown in FIG. 18
(e.g., hemodynamic conditions) on EC and the cardiovascular system (e.g.,
coronary arteries).

[0466]The present example (e.g., FIG. 46) is provided to demonstrate the
capability and utility of the embodiments of the invention for
reproducing in vivo mammalian hemodynamic conditions in vitro. In
particular aspects, the present example will also demonstrate the
exemplary utility of the equipment for obtaining a in vitro and in vivo
information relating to classes and types of dynamic conditions (e.g.,
FIGS. 17A, 17B and 18). Types of dynamic conditions g(t) that were
measured in the present study include changes in production of NO from
ECs exposed to pathologic (e.g., BAECs -180 SPA, FIG. 10B) hemodynamic
conditions versus production of NO from ECs exposed to normal (e.g.,
BAES, 0 deg SPA, FIG. 10A). Additional types of dynamic conditions which
systems 1, 1001 measured and/or controlled include changes in specimens
12 or tubular structure 1112 hemodynamic conditions monitored for Q(t),
D(t), P(t), pH, temperature viability, (directly) and NO, WSS, CS, SPA
(indirectly).

[0467]The cell culture consisted of primary bovine aortic endothelial
cells (BAECs) obtained from fresh aortas. Briefly, fresh bovine aortas
were obtained and rinsed with cold HBSS and 1% penicillin-streptomycin.
The aorta was cut longitudinally along the intercostal arteries and
formed into a trough. Ten ml of collagenase (e.g., Blendzyme from Roche
Diagnostics Corp.) was placed in the trough for 40 min, removed, and
centrifuged (e.g., repeated five times). The cell population purity was
97%-99% as determined via labeled Dil-acetylated LDL, a common marker for
endothelial cells, and flow cytometry.

[0469]Nitric oxide (NO) measurement was performed via a fluorometric
method. Indirect determination was performed via examination of NO
breakdown products NO3.sup.- and NO2.sup.-. The fluorometric
quantification is based on the reaction of nitrite with
2,3-diaminonapthalene (DAN) that produces the fluorescent compound
1-(H)-napthotriazole and can detect concentrations as low as 10 nM. See,
for example, Stamler, J. S., 1995, "S-Nitrosothiols and the Bloregulatory
Actions of Nitrogen Oxides Through Reactions With Thiol Groups," Curr.
Top. Microbiol. Immunol., 196, pp. 19-36, the contents of which are
incorporated herein by reference. Next, 10 μl of DAN solution (0.05
mg/ml in 0.62 M HCl) was added to each well and refrigerated at 4°
C. for 10 min and the reaction was terminated with 10 μl of 2.8 M
NaOH. The fluorometer utilized filters for excitation at 360 nm and
emission at 425 nm (e.g., Packard Fluorocount fluorometer and PLATE
READER Version 3.0 software). Nitrite standards were made with the same
experimental media, phenol red free MEM with 1% BSA+9.5% dextran, in the
range 60 nM-8 μM. The NO concentration range was ˜0.5-3 μM.

[0470]A two-factor analysis of variance model was used with the Tukey
method on a 95% confidence interval. The standardized residuals and
normal probability plot of residuals satisfied model requirements for
linearity (e.g., statistics software from MINITAB®).

[0471]Embodiments of the systems 1, 1101 can include the steady flow
component entering (upstream) the test section where the upstream,
downstream, and external pressures are modulated to impose an oscillatory
component on the steady flow component that resulted in controlled
pulsatile conditions, as shown in FIGS. 10A and 10B. As discussed above,
by appropriate control of these three pressures (types of dynamic
condition shown in FIG. 17), a wide variety of classes of dynamic
conditions (here hemodynamic conditions) can be simulated. FIGS. 10A and
10B show flow, pressure and diameter variation, and 0 deg and -180 deg
SPA, which may be referred to as normal and pathologic hemodynamics from
time to tune. In this study, specimen holder 10 was multiplexed to
accommodate six tubes with individual media lines each including
real-time monitoring and visualization of flow, pressure, and diameter
waveforms therein via a data acquisition system and software. Flow
measurement utilized a noninvasive Doppler ultrasound probe and flow
meter (flow meter model T110 from Transonics®). Pressure measurement
was via an invasive catheter pressure sensor (MPC-500 from Millar®).
Noninvasive inner diameter monitoring required an ultrasound system that
utilized a 10 MHz transducer, pulser/receiver, and 50 MHz high-frequency
data acquisition card (compulite 1250 from GAGE® Applied
Technologies). Sensor signals were acquired in real time with custom data
acquisition software written in LABVIEW® and utilized a DAQ card (400
kHz, PCl-6024E from National Instruments®). Waveform data was analyzed
for desired time periods (e.g., 1 min) and an FFT analysis was performed
to determine functions such as waveform phase angle differences,
magnitude and frequency, calibration scaling, peak max/min, autoscale,
sample acquisition rate. Time lags between DAQ cards, sensors, CPU/BUS,
and software were assessed via an external function generator. The flow,
pressure, and diameter measurements were calibrated from the mass flow
rate, a pneumatic transducer tester (DPM-1B, BIO-TEK® Instruments),
and a precision fabricated tube. All sensors were robust except for the
pressure sensor that would require calibration prior to each experiment.

[0472]PO2/PCO2 control was necessary to ensure proper pH and gas
concentrations for biological experiments. A pH system accommodated six
pH probes (e.g., one per tube) that are multiplexed with a pH meter.
PO2/PCO2 was measured with a blood gas analyzer (CDI300
blood/gas analyzer from Terumo). Temperature was controlled at 37°
C. via a hot plate and large water bath that was enclosed in a thermal
hood. Cell viability was assessed from direct microscope visualization
through an intact tube as well as en face staining (slicing the tube
open).

[0473]The tubular structures 1112 required characteristics of
noncytotoxicity, optical transparency for microscope visualization, and
mechanical properties (e.g., verified using longitudinal stiffness,
KL, where KL/A=ΔF/ΔL/L/A:F is force, A is
cross-sectional area, and L is length) allowing physiologic diameter
variation (±4%) under pressures of 70±20 mmHg. The silicone
elastomer (Sylgard® 184, Dow Corning) was used to fabricate the tubes
or tubular structures 1112, which in this case were six cubes of 8 mm
inner diameter×15 cm length and wall thicknesses of 500 μm.

[0474]Results

[0475]One embodiment of the controller 1103 can produce time varying
control signals fj(t) (e.g., 0 deg SPA and -180 deg SPA) based on
input information fj(t) including specimen size, fluid moving
capacity (e.g., pump size) and location, and desired dynamic conditions
at or along region A or specimen 12 for pressure flow loop subsystem 1105
components. One exemplary method (e.g., controller 1103) will now be
described.

[0476]In this experiment, theoretical approaches for WSS characterization
in straight elastic tubes used sinusoids to approximate prominent
characteristics of physiologic waveforms and to allow emphasis on the
SPA. Alternatively, in vivo measurements of WSS distribution can be used.
See, for example, Shung, K. K., Smith, M. B., and Tsui, B. M. W., 1992,
Principles of Medical Imaging, Academic, San Diego, the contents of which
are incorporated herein by reference. Note that physiologic waveforms
with multiple harmonics cannot be characterized by a single value of SPA.
The calculation of WSS for pulsatile flow in a rigid tube is known as
Womersley's solution. The wall motion in an elastic tube imposes a radial
convective component that affects the WSS. The nonlinear, elastic tube
problem was solved by a perturbation technique that produced correction
factors for Womersley's solution. The corrected pulsatile WSS component
is then added to the steady flow WSS component that can be determined
from a correction factor applied to Poiseuille flow. See, for example,
Womersley, J. R., 1955, "Method for Calculation of Velocity, Rate of Flow
and Viscous Drag in Arteries When the Pressure Gradlent is Known," J.
Physiol. (London), 127, pp. 553-563; Wang, D. M., and Tarbell, J. M.,
1992, "Nonlinear Analysis of Flow in an Elastic Tube (Artery): Steady
Streaming Effects," J. Fluid Mech., 239, pp. 341-358; Wang, D. M., and
Tarbell, J. M., 1995, "Nonlinear Analysis of Oscillatory Flow, With a
Nonzero Mean, in an Elastic Tube (Artery)," J. Biomech. Eng., 117(1), pp.
127-135, the contents of which are incorporated herein by reference.

[0477]Thus, the WSS solution depends on the phase angle (SPA), α,
and the Q, P, and D (e.g., CS) waveforms, which can be provided as input
information fj(t) to controller 1103, which then can determine time
varying control signals fj(t) for a selected embodiment of pressure
flow loop subsystem 1105 components as described below. The waveforms are
decomposed into mean and oscillatory components, where mean components
are defined with a single overbar and oscillatory (sinusoidal) components
are defined with a double overba

WSS= WSS± WSS (1)

Q= Q± Q (2)

D= D± D= D±ε (3)

WSS= WSSpois( Q, D) C( Q,ε,φ± WSSworm( Q, D) C(
Q, Q,ε,φ) (4)

where WSSpois is the mean WSS determined from Poiseuille flow;
WSSworm is the oscillatory WSS determined from Womersley's solution;
C is the correction factor for the mean component [37]; C is the
correction factor for the oscillatory component [38]; ε is the
amplitude of diameter variation; φ is the SPA. The terms in Eq. (4)
are functions of the parameters in parentheses [i.e., for WSSpois(
Q, D), WSSpois is a function Q and D]. The correction factors for
the experimental conditions shown in FIG. 3(A) (0 deg) and FIG. 3(B)
(-180 deg) are

C(500,0.04,0 deg)=0.993,

C(500,0.04,-180 deg)=1.007,

C(500,700.0.04,0 deg)=0.83,

C(500,700.0.04,-180 deg)=1.23

[0478]The resulting WSS waveforms are WSS=10±10 dyne/cm2 for both
cases. Note that the correction factors for these experimental conditions
indicate that for SPA=-180 deg, the flow amplitude, Q, should be 23%
lower than the Womersley flow amplitude, and at SPA=0 deg, Q should be
17% larger than the Womersley flow amplitude. The correction factors for
the mean components were negligible for these conditions.

[0479]Feedback information FBj(t) can be determined in the selected
embodiments of pressure flow loop subsystem 1105 components and output to
controller 1103 to assess conditions in system 1101 (e.g., at region A or
along conduit 3701) In this study, FBj(t) included at least Q(t),
D(t), P(t), pH, temperature, viability and NO, WSS, CS, SPA.

[0480]In this study, long-term stability of the system under pulsatile
conditions was assessed via continuous monitoring of the Q, P, and V
waveforms over a 36 hr period at 37 deg, which showed controlled
maintenance of the dynamic conditions or waveforms. PO2/PCO2
concentrations were measured after pulsatile conditions with a blood gas
analyzer and were shown to have similar values to incubator controls of
the same time duration of 17 h, PO2=141 mmHg and PCO2=40 mmHg.
The temperature was very stable (±0.5° C.) and was not affected
by opening/closing the thermal hood door. In this study, there were minor
variations in the operating conditions over the 15 cm tube length in the
test section. The variations across the tube length (L) during pulsatile
flow at SPA=0 deg and -180 deg were: ΔP=1.5-2 mmHg, ΔQ=10
ml/min, Δτ=0.2 dyne/cm2 (calculated), and ΔSPA=26
deg.

[0481]FIG. 50 shows production of NO from BAECs exposed to hemodynamic
conditions in media at 5 hr. In FIG. 50, pairwise significant differences
indicated by * for 0 deg SPA and -180 deg SPA, # for 0 deg SPA and steady
state (SS), and ** for dynamic and static controls with p values <0.05
(n=5). The biological results in this study depicted in FIG. 46 show a
significant decrease in NO quantity for the pathologic -180 deg SPA
versus the normal 0 deg SPA case (p<0.05). The 0 deg SPA case was
significantly higher than the steady shear (SS) case (p<0.05). All the
dynamic conditions were significantly higher than the static cases,
static control (SC), and pressurized control (PC) (p<0.05). The SS
case verified that the endothelial cells exhibited the anticipated
increased NO shear response compared to the SC case. The PC case was not
significantly greater than the SC case.

[0482]Systems 1, 1101 simulate normal and pathologic hemodynamics (e.g.,
FIG. 46). Complex physiologic hemodynamic features associated with
different vascular beds can be simulated in vitro. In this embodiment the
system utilized three-dimensional geometries (i.e., silicone tubes) to
provide a physiologic environment to control or systematically evaluate
or model concomitant influences of Q, P, and D waveforms on vascular
physiology and/or pathology (e.g., fluid molecules such as gene and
protein expression profiles).

[0483]As shown in FIG. 50, the pressurized control (PC) case was not
significantly increased compared to the static control (SC) case,
implying that the mean pressure and circumferential strain (CS) do nor
have a significant influence on NO production compared to steady shear
stress. However, concomitant SS and CS affected the NO response of
endothelial cells, modulated by the SPA. The significantly lower NO
response of the -180 deg versus the 0 deg SPA case along with companion
controls indicated that the large negative SPA had a negative or
pathologic effect on the NO response. Regions of the circulation prone to
pathologic development (i.e., atherosclerosis and intimal hyperplasia),
such as the aortic abdominal bifurcation and curved coronary artery,
experience a highly negative SPA. See, for example, Lee, C. S., and
Tarbell, J. M., 1997, "Wall Shear Rate Distribution in an Abdominal
Aortic Bifurcation Model: Effects of Vessel Compliance and Phase Angle
Between Pressure and Flow Waveforms," J. Biomech. Eng., 119 (3), pp.
333-342; and Qiu, Y., and Tarbell, J. M., 2000, "Numerical Simulation of
Pulsatile Flow in a Compliant Curved Tube Model of a Coronary Artery," J.
Biomech. Eng., 122(1), pp. 77-85; the contents of which are incorporated
herein by reference.

[0484]Although embodiments of the invention have been described with
reference to a number of illustrative embodiments thereof, it should be
understood that numerous other modifications and embodiments can be
devised by those skilled in the art that will fall within the spirit and
scope of the principles of this invention. More particularly, reasonable
variations and modifications are possible in the component parts and/or
arrangements of the subject combination arrangement within the scope of
the foregoing disclosure, the drawings and the appended claims without
departing from the spirit of the invention. In addition to variations and
modifications in the component parts and/or arrangements, alternative
uses will also be apparent to those skilled in the art.